Biological Impacts of Microplastic Exposure in Zebrafish (Danio rerio): A Systematic Review Across Developmental, Physiological, and Neurobehavioral Endpoints

Abstract

Microplastics (MPs) are emerging pollutants widespread in aquatic environments; however, their effects across the different life stages of aquatic organisms remain poorly understood. This systematic review integrates recent experimental results on the developmental, physiological, and neurobehavioral effects of MP exposure on zebrafish (Danio rerio), a popular model organism for ecotoxicology research. A PRISMA-guided search using Web of Science (WoS) and Scopus as databases generated 581 articles, which were screened to 60 eligible articles. The collated results showed that MP toxicity at various life stages of zebrafish was strongly related to the physicochemical properties of MPs and exposure conditions. In terms of developmental toxicity, peer-reviewed publications assessing specific MP physicochemical properties—polymer type, size, concentration, shape, and degree of aging—reported concentration-dependent effects, with increasing MP concentrations generally associated with growth inhibition, cardiac dysfunction, increased malformations, and lower hatching rate, particularly at ≥10 mg/L to ≥100 mg/L. However, several studies noted that under particle-based exposure scenarios, MP toxicity exhibited threshold-like or non-monotonic responses, attributed to aggregation, bioavailability, and uptake dynamics. Weathered and artificially aged MPs exhibited higher embryotoxicity and neurodevelopmental toxicity, including changes in gene expression of neurons, decreased integrity of motor neurons, and impaired retinal development, compared with “virgin” MPs. In terms of physiological endpoints, oxidative imbalance like changes in the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)), lipid peroxidation, inflammation, and disruption of tight junctions have been reported as key toxicity pathways. Chronic MP exposure in zebrafish also caused changes in the gut microbiota, hepatic metabolism, endocrine disruption, reproductive damage, thyroid function disruption, and genotoxicity in zebrafish. In terms of neurobehavioral effects, changes in locomotor activity, anxiety response, neurotransmitter homeostasis, and acetylcholinesterase function, have been observed, in both larvae and adults, with a potentiation effect in aged MP exposure. Finally, this systematic review found major limitations for inter-study comparisons because of inconsistencies and differences in methodology applied related to MP concentration, simulation of natural MP aging, and MP dose measurements.

1. Introduction

Microplastics (MPs) are emerging pollutants that have recently become ubiquitous in the environment due to the rapid increase in global plastic production, widespread adoption of plastic-based materials for packaging of fast food and online delivery services, the “sachet culture” predominant in low and middle-income countries consumption, fast fashion utilizing plastic fibers like polyester, and underdeveloped municipal solid waste management infrastructure in the majority of developing countries [1,2]. According to Dokl et al. [3] global plastic consumption is forecasted to increase from 464 million tons in 2020 up to 884 million tons by 2050 (Figure 1). Unfortunately, only a small portion of plastic waste generated globally, about 10%, will be collected, incinerated or properly disposed of in landfills while ~90% ended up in watersheds, rivers, water bodies, and the ocean [4]. According to the European Food Safety Authority (EFSA) [5], MPs are heterogenous mixtures of materials consisting of synthetic polymer particles, films and fibers with dimensions ranging from 0.1 µm to 5 mm. These pollutants generally vary in shape, composition, density, and degree of environmental weathering, influencing their environmental fate and biological impacts [6]. Microplastic pollution is commonly prevalent in bodies of water in metropolitan and urban areas with high human population density [7]. In surface water samples from the Pasig River in Metropolitan Manila, Philippines were found to be polluted with MPs (63,000 MP particles/km²), contributing to the ecotoxicological burden in not only the river but also the Manila Bay [8]. The population density in the Pasig River basin is around 21,000 persons/km², and the MPs identified in the Pasig River include polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl chloride (PVC), polyacrylamide (PAM), and polyurethane (PU) [9]. Fish stocks sourced from water bodies with large population density have also been reported to contain MPs. In the Yangtze River Basin in China with a population of ~459 million, for example, 80% of fishes examined had various types of MPs in their digestive system, including PE, PP, and polyethylene terephthalate (PET) [10]. Similarly, up to 28.4% of commercially imported fishes from European coastal waters were found contaminated with MP fragments smaller than 1 µm [11].

Microplastics are formed in the environment either from the disintegration of large plastics due to prolonged exposure to ultraviolet (UV) radiation, mechanical abrasion, and weathering processes [12] or the inadvertent release of manufactured plastic microbeads found in skin care products and abrasives [13]. Anthropogenic activities, including waste disposal and transportation, textile manufacturing, construction projects, and medical wastes, are major contributors to MP pollution [14]. The textile industry, for example, contributes to MP pollution through the production of plastic fibers such as polyester, nylon, and acrylic, their processing into textiles for carpets and clothing, and the repetitive washing of these materials [15]. Transportation is another sector that contributes to MP pollution, mainly through the abrasion of tires on roads, which are dispersed in the air or washed away into aquatic ecosystems through runoff and the sewerage systems [16]. Meanwhile, the construction industry contributes to MP pollution due to the degradation of synthetic paints, polymer-based coatings, and plastic insulations, which are then washed away into the aquatic ecosystem through runoffs [17]. Other sources of MP pollution include the degradation of plastic packaging, disintegration of plastic fishing gears, fragmentation of plastic films, and the deposition of synthetic fibers from urban emissions [18].

Due to their small size and hydrophobic properties, MPs have become widespread across diverse environments. They can be easily transported via air and water, and bioaccumulate through the food chain, affecting all living organisms including human population [19]. Moreover, the widespread occurrence of MPs in the aquatic ecosystems indicates that aquatic life, including fishes, are continuously exposed to different levels of MP contaminations [20]. Barboza et al. [13], for example, reported that fish gastrointestinal tract usually held the highest proportion of total MPs at about 40%. This pattern of accumulation has wider implications than just fish health, as seafood is a major dietary source of protein for the majority of human populations, creating a potential exposure pathway for consumers [21]. Some estimates have placed MPs ingested by adults annually via seafood as high as 842 MPs particles, with predicted higher intakes in areas where fish stocks exhibited greater contamination levels [12]. Once ingested, MP particles have been shown to translocate across biological membranes, leading to bioaccumulation in the human body, which may initiate cellular stress responses and inflammatory reactions [22]. Laboratory experiments also suggest that MPs may cause oxidative stress, cytotoxicity, and immune responses in not only model organisms but also human cells due to the high reactivity of the MP surfaces, as well as the leaching of plastic additives such as bisphenols and phthalates [23,24].

Among aquatic model organisms, zebrafish (Danio rerio) has been widely used in ecotoxicology studies due to its conserved vertebrate organ systems, including the liver, intestine, endocrine system, immune system, and reproductive organs, enabling detailed physiological response assessment, such as oxidative stress, inflammation, metabolic disruption, endocrine dysfunction, and histopathological alterations [25,26]. The transparent embryos of zebrafish also facilitate direct visualization of abnormal development during embryogenesis and organogenesis, hatching success, cardiac development, and morphological abnormalities, thus, providing precise evaluation of the teratogenic effects of environmental contaminants [27]. The rapid development of zebrafish embryos and their high reproductive capacity make them suitable for conducting toxicity tests on a large scale in the laboratory [28]. Furthermore, zebrafish exhibit well-characterized behavioral repertoires and neurotransmitter systems that allow the quantification of locomotor activity, anxiety-like behavior, predator avoidance, learning responses, and neurochemical alterations following contaminant exposure [29,30,31]. Moreover, the genetic similarity of zebrafish to humans is very high because 70% of the human genome contains at least one ortholog in the zebrafish genome [31]. These characteristics of zebrafish makes it an excellent animal model for studying the effects of environmental contaminants, including MPs [26].

The use of zebrafish was first considered in studying anticancer therapy [18]. As their genetic makeup and phenotypic traits became better characterized, their utilization for research expanded, leading to widespread applications in diverse research fields, including biomedical and toxicological studies. The rapid acceptance of zebrafish in animal studies was further justified by their practical and biological advantages, including low breeding costs, straightforward husbandry, and simple ethical frameworks that facilitated their use across diverse study methods [32].

Previous studies on the short-term ecotoxicity of PS MPs using zebrafish showed that tissue distribution, and organ-specific effects like hepatic alterations correlated with MP particles size of 5 μm [15]. MP exposure has also been linked to intestinal injury phenotypes in zebrafish, as well as oxidative stress related effects in controlled experiments that investigated multiple types of MPs including PE, PS, PP, PVC, PET, polyethersulfone (PES), polylactic acid (PLA), polyamide (PA), polyhydroxyalkanoates (PHA), and polyglycolic acid (PGA) [33]. In modern molecular toxicology, oxidative response is defined as the first line molecular signal for toxic stimuli exposition [34]. Although the modulatory activity of oxidative/reducing pathways and reactive oxygen species (ROS) is responsible for oxidative balance, disturbances in such processing of harmful stimuli, caused by unusual dysregulated or disordered in metabolism of ROS, could be a significant source of cellular toxicity [35,36].

Neurobehavioral and neurochemical alterations have been reported in larval zebrafish after exposure to virgin and aged PS MPs, including changes in locomotor activity and neurotransmission-related parameters [37]. At the molecular level, transcriptomics has demonstrated the possibility for muti-system impacts of MP exposure, which caused broad gene expression changes, developmental, and neurobehavioral effects in zebrafish [38]. Aging and weathering alter MP morphology, surface chemistry, and toxicity, making it important to differentiate pristine from environmentally weathered MPs when constructing zebrafish hazard evidence [39]. In the study of Mansuri et al. [39] that compared the effects of weathered and pristine MPs, the results indicated significantly higher mortality in weathered MPs (80%) compared with virgin MPs (20%). In zebrafish larvae, MP ingestion promoted disruptions of key molecular pathways, including oxidative stress response, apoptosis, and DNA damage repair [39]. Similarly, evidence in adult zebrafish showed MPs exposure could co-occur with immune-related and behavioral effects based on transcriptional changes, suggesting the importance and interdependence of developmental, physiological, and neurobehavioral outcomes [15]. Luan et al. [32] reported that PGA MPs exposure of zebrafish at 100 mg/L significantly decreased the survival and hatching rates at 10 and 24 h post-fertilization (hpf), increased wakefulness, reduced sleep in 80% of zebrafish. Similarly, 100 mg/L of PLA MP exposure and at 5 Days post-fertilization (dpf) exhibited impaired neurobehavior by decreasing voluntary locomotor activity, inducing anxiety-like behavior, and disrupting the circadian sleep-wake cycle, characterized by increased wakefulness and reduced sleep duration, indicating disruption of circadian rhythm.

Recent review papers have addressed the potential of model organisms, especially zebrafish, to evaluate the ecotoxicological effects of MPs and other emerging microcontaminants [27,28]. Several review articles have also discussed the ecotoxicological effects of MPs in aquatic organisms, including zebrafish as a model species, but the majority have focused on specific aspects of MP toxicity, such as oxidative stress, reproductive toxicity, molecular responses, or environmental occurrence. Meanwhile, other reviews have provided broad overviews of MP pollution and its biological consequences but did not systematically integrated evidence across multiple biological systems within zebrafish. In contrast, this systematic review synthesized developmental, physiological, and neurobehavioral endpoints within a single analytical framework and evaluated how toxicity patterns varied according to MP properties, including polymer type, particle size, shape, degree of weathering or aging, and extent of exposure (i.e., concentration). By integrating evidence across different life stages and biological responses, this review provides a more comprehensive assessment of the biological consequences of MP exposure in zebrafish and identified methodological limitations that constrained inter-study comparisons and ecological risk assessment.

A structured and systematic search of Scopus and Web of Science (WoS) databases was performed using the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) 2020 [40] (Table S1) to identify experimental studies assessing the biological impacts of different MP types, sizes, concentrations, and exposure times on zebrafish. The systematic review combined the results from different life stages, including embryos, larvae, juveniles, and adults, to provide a comprehensive evaluation of biological impacts. The current study aims to answer the following research questions:

i.

How does exposure of zebrafish to MPs affect their developmental, physiological, and neurobehavioral endpoints in relation to the underlying toxicity mechanisms, dose-response relationships, and severity of biological impacts?

ii.

What are the key methodological constraints, exposure variables (such as polymer type, particle size, concentration, and degree of weathering), and inconsistencies in experimental studies that limit the interpretation of biological impacts and risk assessment in zebrafish models?

2. Materials and Methods

2.1. Search Criteria

The Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) 2020 guidelines were employed for article identification, screening, eligibility assessment, and inclusion/exclusion criteria. Prior to the search, the protocol was pre-registered in the Open Science Framework (OSF) with registration DOI: 10.17605/OSF.IO/KHMJA. The search was conducted on August 2025 using Web of Science (WoS) and Scopus databases. The search strategy employed the terms “microplastic” AND “zebrafish” to identify studies specifically investigating the biological effects of MP exposure in zebrafish. The search was restricted to articles published between 2015 and 2024. The findings of this review should be interpreted within the scope of the selected databases and search terminologies. Also, the search strategy was designated to prioritize specificity and direct relevance to zebrafish-based MP toxicity studies rather than maximize retrieval breadth. The specific search string used was “TITLE-ABS-KEY (zebrafish AND microplastic) AND PUBYEAR > 2014 AND PUBYEAR < 2025”.

2.2. Selection Process (Inclusion and Exclusion Criteria)

The initial search of the two databases yielded 581 items. Following the removal of duplicates, 371 articles were filtered according to document type and language. Only peer-reviewed research articles published in English were included. Articles classified as reviews (60), non-English (8), conference (1), proceedings (1), editorials (1), letters and notes (2), erratum (1), and retraction (1) were excluded (Figure 2). Further filtering of the title and abstract were done to identify relevance to experimental studies investigating MP exposure in zebrafish. Studies not conducted on zebrafish, not investigating MP exposure, or not evaluating biological endpoints were also excluded.

Following title and abstract screening, potentially eligible studies underwent full-text assessment based on the predefined inclusion and exclusion criteria, reducing the final eligible articles to 60. For each eligible article, information was extracted regarding MP properties (i.e., polymer type, particle size, shape, and extent of weathering), dosage (i.e., concentration), zebrafish life stage, exposure duration, biological endpoints assessed, and major toxicological outcomes. Due to differences in experimental design, exposure metrics, and endpoint measurements among studies, a quantitative meta-analysis was not feasible. Therefore, the evidence was synthesized narratively by grouping studies according to developmental, physiological, and neurobehavioral endpoints and identifying common toxicity patterns.

2.3. SYRCLE Risk of Bias Assessment

The 60 eligible studies were evaluated using the SYRCLE Risk of Bias tool, and the results are summarized in Figure 3. The assessment results suggest that studies included in the review were characterized by moderate methodological quality, with most domains demonstrating a predominance of low-risk judgments. Several domains also remained classified as having an unclear risk of bias, indicating that methodological reporting was frequently incomplete, which limited confidence in the assessment of study quality.

Domains 8, 9, 12, 13, 18, and 19 consistently exhibited a greater proportion of low-risk judgments than either high- or unclear-risk assessments. This pattern suggests that the majority of studies adequately reported important methodological components evaluated by the SYRCLE tool, reflecting relatively consistent implementation of these aspects across the experimental literature. In particular, the high proportion of low-risk assessments in Domains 12 and 13 indicates that these methodological elements were generally well addressed by the eligible studies.

In comparison, uncertainty in the risk-of-bias assessment was concentrated within several domains, particularly Domains 4, 6, 14–17, 19, and 20, where unclear judgments accounted for a substantial proportion of the included studies. These results likely reflected insufficient methodological reporting rather than demonstrable methodological deficiencies. In many studies, essential information required to evaluate these domains was either incompletely described or omitted entirely, preventing a definitive judgment regarding the presence or absence of bias. These reporting limitations are frequently encountered in animal toxicology studies and reduce the transparency and reproducibility of experimental findings.

High-risk judgments were comparatively less common across the assessment. However, Domains 5 and 7 showed a relatively greater proportion of studies classified as high risk, while Domains 1, 3, 6, 11, and 20 demonstrated notable frequencies of high-risk assessments. This distribution suggests that certain methodological practices evaluated by these domains were implemented inconsistently across the eligible studies or were associated with identifiable sources of bias that could potentially influence experimental outcomes. Although methodological weaknesses were not pervasive throughout the evidence base, they remained evident within specific components of study design and conduct.

2.4. Bibliometric, Co-Occurrence, Author Coupling Analyses and Geographic Distribution of Published Studies

Using VOSviewer version 1.6.20, an analysis of keyword co-occurrence and bibliographic coupling was carried out to visualize the intellectual structure of the literature related to MP toxicity in zebrafish [41]. This analysis identified frequently occurring keywords and research clusters that define the current scope of MP-related biological research in zebrafish. As illustrated in Figure 4, dominant keywords showing the strongest connections included “microplastics”, “zebrafish”, and “toxicity”. These terms showed strong connections with frequently used keywords, such as “oxidative stress”, “development”, “neurobehavior”, “accumulation”, and “gene expression”, which are the primary endpoints of MP-related zebrafish studies. Moreover, the bibliographic coupling and co-authorship network analysis revealed authors who contributed significantly to the field of MP toxicity research in zebrafish (Figure 5). In Figure 5, node size reflects the frequency of keyword occurrence, while the connecting lines indicate the strength of co-occurrence between terms. The color gradient represents the average publication year, with blue indicating earlier studies and yellow representing more recent research trends.

The network is centered on the keyword’s “zebrafish” and “microplastics”, indicating that these are the dominant research themes and serve as the primary hubs linking multiple areas of investigation. Frequently associated terms include oxidative stress, apoptosis, microbiome, metabolomics, neurotoxicity, developmental toxicity, and gene expression, highlighting that current zebrafish studies predominantly examine the molecular and physiological mechanisms underlying microplastic-induced toxicity. The authors who showed the strongest links are the ones whose publications showed a large number of references to other publications within the dataset, showing the strong intellectual connections within the research community. These clusters of authors demonstrate the collaborative nature of MP toxicity research, where some authors have contributed significantly to the understanding of the impact of MPs on the development, physiology, and neurobehavior of zebrafish. Lastly, Figure 6 presents the geographic distribution of published studies investigating the biological impact of MP exposure in zebrafish between 2015 and 2024. Countries are color-coded according to the number of studies they contributed, with warmer colors indicating greater research output. The star symbol denotes the country with the highest research output among all countries. China (Mainland) was the dominant contributor with 34 studies, followed by India (7), South Korea (5), and Portugal (4). Greece, Brazil, and Singapore each contributed 2 studies, while Norway, France, Italy, and Oman each contributed 1 study.

3. Developmental Anomalies and Consequences of Microplastic Exposure in Zebrafish

Embryonic and larval exposure to MPs demonstrated clear concentration-dependent developmental toxicity. Higher concentrations of polystyrene (PS) with a particle size of 1–5 μm, at 10 µg/L, showed no significant effects on survival or hatching and a minor to non-significant increase in pericardial edema (5%). At 100 µg/L, the hatching rate was reduced to 10%, and there was a slight increase in tail curvature (8–10%). PS MP exposure at 1000 µg/L significantly delayed hatching to -25%, heart rate to -13% vs. control, and increased embryo deformities up to 22% (Figure 7) [42]. However, certain particle-based exposures revealed non-linear dose responses, with moderate particle loads producing measurable cardiac and growth suppression while higher mass-based concentrations did not proportionally increase embryotoxicity [37]. For instance, particle aggregation and bioavailability have been found to decrease with increased concentrations in zebrafish studies where MPs were found to cluster in a way that reduced their interaction with embryos [38]. In addition, saturation of uptake pathways and limits to internal particle accumulation have also been described in zebrafish experiments where increasing exposure concentrations did not proportionally increase internal particle burden or toxicity, indicating biological thresholds in uptake or transport processes (Table 1) [43].

Low-dose exposure to PS, PE, and PES frequently showed no mortality and significant change in body length and survival, as reported by Medriano and Bae [43], Luo et al. [44], and Yang et al. [45]. Developmental toxicity increases with concentration but may exhibit non-monotonic or threshold-like patterns depending on particle metrics. For example, exposure to fragmented PE and PS MPs at 0.2 mg/L and 1 mg/L produced no significant differences in survival, cardiac endpoints, or developmental parameters, indicating a plateau response where the increasing concentration did not correspond to increased toxicity [46]. Similarly, experiments using particle-based exposure metrics for artificially weathered polypropylene and polystyrene showed that moderate particle loads (2000–20,000 MP·L⁻¹) resulted in reduced heart rate and body length in embryos, whereas a much higher exposure level (200,000 MP·L⁻¹) did not further intensify embryotoxic effects, suggesting a non-linear relationship between particle abundance and developmental outcomes [38]. Another example is observed in PS microbead exposure experiments where embryos exposed to 10 µg/L showed no significant developmental effects, while intermediate concentrations such as 100 µg/L produced measurable reductions in hatching and mild deformities, and extremely high concentrations (1000 µg/L) caused developmental delay and increased malformations, demonstrating a threshold-like transition from negligible to pronounced toxicity as the concentration increased [42]. These findings indicate that developmental responses to MPs are not always proportional to concentration and may depend on particle number, size distribution, and physicochemical interactions within the exposure system.

Furthermore, particle aging significantly amplified developmental and neurodevelopmental toxicity. Photo-aged and weathered PS and PE MPs consistently resulted to (~80–82%) compared with virgin PS particles, which produced only ~10–20% mortality and were not significantly different from controls. In the same study, weathered MP treatments also produced markedly higher malformation rates, including spinal deformities (~13–14%) and bent tails (~21%), whereas virgin PS exposures resulted in low or statistically insignificant malformation frequencies [39]. Similarly, photo-aged PS particles (1 µm) produced stronger growth inhibition and survival reduction in zebrafish larvae compared with virgin PS at equivalent concentrations (0.1–100 µg/L), indicating enhanced toxicity after environmental aging [47]. Another study examining UV-aged PA MPs demonstrated clear concentration-dependent growth impairment: exposure to 10 µg/L caused slight growth inhibition, 100 µg/L significantly reduced larval body length and induced intestinal structural damage, and 1000 µg/L resulted in severe growth inhibition accompanied by pronounced intestinal damage and impaired lipid absorption. In this experiment, photo-aged PA particles produced significantly stronger toxic effects than pristine particles across all exposure levels (Figure 8) [48]. Artificially aged PS reduced motor neuron fluorescence and dysregulated neurodevelopment-related genes including gabra1, manf, nestin, and gfap, indicating interference with neuronal differentiation and central nervous system formation [49]. Retinal exposure to fluorescent microspheres altered proliferation markers and downregulated neurogenesis genes such as sox2 and neuroD, suggesting compensatory but disrupted neurodevelopmental signaling [50]. The enhanced toxicity observed in aged or weathered MPs is primarily attributed to physicochemical transformations that occur during environmental aging processes such as ultraviolet (UV) irradiation, oxidative degradation, and mechanical abrasion. These processes introduce oxygen-containing functional groups (e.g., carbonyl and hydroxyl groups) on the polymer surface, increasing surface polarity and reactivity, which enhances interactions between particles and biological membranes (Figure 8) [51,52]. Another mechanism contributing to enhanced toxicity is the increased ability of weathered MPs to adsorb environmental contaminants, including heavy metals, hydrophobic organic pollutants, and plastic additives. These adsorbed compounds can be transferred to organisms during exposure, allowing aged MPs to act as vectors of secondary toxicants and amplifying biological effects [51,53]. In zebrafish models, these combined effects can stimulate excessive ROS production and disrupt antioxidant defense systems, leading to oxidative stress, inflammation, and altered expression of genes involved in neurodevelopmental processes [18]. Additionally, polymer type and particle size further influenced developmental outcomes. For instance, exposure to PVC MPs with a mean particle size of approximately 250 µm at concentrations of 100, 200, 300, and 400 ppm showed increasing developmental abnormalities in zebrafish embryos. While survival and hatching were not significantly affected across these exposure levels, significant edema formation was observed at the highest concentration (400 ppm) by 120 hpf, and tail malformation rates reached up to ~19.1% in the 300–400 ppm exposure groups, indicating concentration-dependent teratogenic effects [54]. In contrast, fragmented PE and PES MPs with average sizes of ~180 ± 210 µm (PE) and ~350 ± 220 µm (PES) produced minimal developmental toxicity at lower concentrations. Acute exposure experiments using 0.2 mg/L and 1 mg/L of these particles showed no significant differences in survival, cardiac parameters, or major developmental abnormalities, suggesting that relatively large fragmented particles at low concentrations exert limited embryotoxic effects under short-term exposure conditions [46,55].

Microbead PS at 10 mg/L resulted in measurable malformation and mortality across different size classes, demonstrating that even microscale particles can induce structural abnormalities in zebrafish embryos [56]. In one study, exposure to 5 ± 3 μm PS microbeads produced 31.3% malformation and 11.1% mortality, with 72 hpf hatching at 72.2% and 96 hpf hatching at 87.5%. A similar experiment using larger 50 ± 3 μm PS microbeads at the same concentration resulted in 27.78% malformation and 8.3% mortality, with 68.0% hatching at 72 hpf and 88.9% at 96 hpf, indicating that smaller particles produced slightly stronger developmental effects under equivalent exposure conditions [56]. Additional studies further demonstrate the influence of particle size on developmental toxicity. For example, exposure to 1 and 5 μm PS particles at concentrations of 0.1, 1.0, and 10.0 mg/L showed increasing embryotoxicity with both concentration and particle size characteristics. At 10.0 mg/L, zebrafish embryos exhibited an approximately 25% reduction in hatching rate, a 13% decrease in heart rate, and malformations in about 22% of embryos, including pericardial edema and tail deformities [37].

Lastly, growth endpoints at various stages of life reveal that chronic exposure refers to longer experimental durations ranging from 7 days to several weeks (e.g., 14–60 days) and frequently involves higher cumulative exposure levels (Table 1). Chronic studies commonly used concentrations between 1 mg/L and 100 mg/L, with stronger physiological and developmental effects observed at the upper end of this range. For example, long-term exposure to ≥10 mg/L PE MPs resulted in a reduced body length, metabolic disruption, and increased oxidative stress biomarkers, while very high chronic exposures such as 50–100 mg/L produced pronounced physiological damage, including hepatic injury and oxidative imbalance in zebrafish tissues [57,58]. Nevertheless, some low-dose exposures did not present statistically significant differences in growth parameters, reiterating the significance of concentration thresholds. In several zebrafish experiments, these thresholds appear to occur at relatively low exposure ranges. For example, exposure to spherical PS of ~5 µm at 20 µg/L for 21 days showed no significant changes in body length, body weight, or survival, indicating that concentrations at or below this level may fall below the threshold required to induce measurable growth impairment under chronic exposure conditions [44]. These thresholds are not universal and vary depending on the polymer type, particle size, and particle morphology. Polymer composition influences toxicity because different plastics contain distinct additives and surface chemistries that affect particle reactivity and interaction with biological tissues. For instance, PVC MPs produced developmental abnormalities at high concentrations (300–400 ppm), while PE fragments showed minimal effects at lower mg/L levels, indicating polymer-dependent toxicity differences [54,59].

Table 1. Developmental/neurodevelopmental effects of pristine/virgin/undisclosed and UV/photo-aged MP exposures.

Life Stage	Plastic Characterization (Type/Shape/Size/Concentrations)	Endpoints	Exposure Time	Effects	Ref.
Adult	PP/Spherical/Virgin: 33.2 ± 14.4 μm/UV: 20.8 ± 10.5 μm/50 mg/L for PP and UV-PP	Survival rate, hatching rate, time to hatch	14 days	Excretion began immediately after MP removal, GI residual after 1 day: PP = 7.9%, UV-PP = 12.3%, elimination half-life: PP = 0.78 days, UV-PP = 0.38 days, 99.9% excreted after 5 days.	[42]
	PE & PES/Fragmented beads/average size of 180 ± 210 µm/1 mg/L both PE & PES	Growth and development: body length and survival rate	96 h (4 days) acute exposure	No significant developmental toxicity.	[43]
	PS/5 µm/2 mg/L	Hatching rate (72 hpf), body length (7 dpf), heart rate, and malformation rate (pericardial edema, yolk sac edema, spinal deformity)	14 days	No recorded mortality for PS MP exposure.	[58]
	PE & PES/Fragments/PE mean size: 180 ± 210 µm PES mean length: 350 ± 220 µm/0.2 mg/L, 1 mg/L both PE & PES	Cardiac and developmental: heart rate, pericardial edema, cardiac looping, blood flow abnormalities, survival and hatching	30 days	No mortality observed, no major difference between 0.2 and 1 mg/L (threshold effect).	[45]
	PS/Spherical beads/5 µm diameter/20 µg/L	Growth indices: body weight, body length, condition factor (K = W/L3 × 100)	21 days	No mortality occurred; final body length and body weight did not change.	[44]
	PGA/~1 μm in diameter/1 mg/L & 100 mg/L	Neurochemistry: brain 5-HT system	28 days	↓ brain 5-HT both 1 mg/L & 100 mg/L. Serotonin-pathway gene expression: ↑ tph1b while ↓ tph1a. Brain inflammatory gene expression: ↑ (il-1β, tnf-α, il-10) after PGA exposure.	[47]
Embryos	PS/Spherical beads/1, 5 µm/10, 100, 1000 µg/L	Survival rate, hatching rate, time to hatch	96 h (4 days post-fertilization)	PS (10 µg/L) No significant effects on survival or hatching. Minor but non-significant increase in pericardial edema (5%). PS (100 µg/L) ↓ Hatching rate (~10% reduction); slight increase in tail curvature (8–10%). PS (1000 µg/L) Significant developmental delay; ↓ hatching (−25%), ↓ heart rate (−13% vs. control), ↑ deformities (22% embryos malformed).	[37]
	Virgin & Photo-aged PS/1 μm diameter/0, 0.1, 1, 10, 100 μg/L for virgin and photo-aged PS MPs	Embryo development and mortality (daily observation); neurotoxicity-focused endpoints: locomotor behavior, neurotransmitter changes, neuronal development and gene expression related to neurotransmission/adipocytokine signaling.	24 h	Motor neuron development (Tg(hb9-GFP), 120 hpf): A-PS exposure reduced motor neurons in brain and spinal cord relative to control; GFP fluorescence intensity decreased from 238 ± 2.58 AU (control) to 234 ± 2.88 (0.1 μg/L), 232 ± 4.97 (1 μg/L), 230 ± 3.95 (10 μg/L), and 229 ± 2.53 AU (100 μg/L) (p < 0.05). Neurodevelopment- and nervous system-related gene expression (120 hpf): A-PS significantly altered expression of nervous system function/development genes (nestin, gfap, manf, shha, alpha–tubulin, mbp). Relative to control, A-PS significantly inhibited gabra1 and manf, and significantly elevated nestin, gfap, alpha–tubulin, and mbp.	[48]
	Virgin & Artificially weathered PP & PS/PS: ~15–36 µm PP: ~50–148 µm; after weathering: (≤230 µm)/Environmentally relevant (particle based): 2000, 20,000, 200,000 MP·L−1; high concentrations (mass based): 12.5, 25, 50, 100 mg·L−1	Mortality, hatching rate	96 h (outcomes recorded at 24/48/72/96 h; heart rate at 72 h; length at 96 h)	2000 MP·L−1: ↓ heart rate, ↓ body length. 20,000 MP·L−1: sublethal growth effects. 200,000 MP·L−1: no linear increase in toxicity. 12.5–100 mg·L−1: no significant embryotoxicity.	[38]
	PS/spherical/0.1 μm diameter/0, 0.1, 1, 10, 50, 100 mg/L	Development: cumulative mortality, hatching, malformation (48–96 hpf); morphometrics: yolk sac area, pericardial edema, body length, eye size, heart rate.	96 h	PS attenuated AgNP toxicity rather than acting as a strong developmental toxicant at the selected dose.	[49]
Embryos → Larvae	Fluorescent plastic microspheres/spherical/1–5 μm/2 mg/L (~1.09 × 108 particles/L)	Developmental and biodistribution: mortality, growth, distribution of MPs in tissues	2 hpf to 14 dpf	Increased PCNA-positive cells in retina → increased cell proliferation, downregulation of neurogenesis genes (sox2, neuroD, olig2), altered DNMT expression (epigenetic modulation), presence of plastic particles in retina.	[50]
	Not disclosed/MPs: ~1 µm diameter/MPs suspended at 0.006%, 0.0045%, 0.003%, 0.0015% solids	Mortality rate, tail and vascular morphology (larval stage), angiogenesis, growth metric (caudal body length), caudal vein morphometrics, and heart morphology	Exposed for 3 days (1 dpf → 4 dpf)	Developmental toxicity included: pathological changes of caudal vein plexus (angiogenesis abnormalities), caudal tissue impairment and reduced growth/body length, peripheral microcirculation dysfunction (caudal region). Mortality (1 dpf → +1day exposure): mortality from 29.2% (MP1) to 95.8% (MP4), with intermediate values 33.3% (MP2) and 58.3% (MP3). Heart morphology: described as largely transformed (malformed) relative to control zebrafish.	[60]
	PS/Fragmented/2 µm/10 mg/L	Developmental toxicity: blood disorder, heartbeat, hatch/death rates, malformations, morphometrics; neurodevelopmental marker: atoh1a expression in cerebellar area (fluorescent reporter)	24–72 hpf	Developmental toxicity: single PS 157 μm showed no observed effect. Neurodevelopmental toxicity: μ-PS did not change cerebellar fluorescence.	[61]
	PGA, PLA, PBS, PHA, PBAT/No data/1 mg/L and 100 mg/L for each polymer	Survival: assessed repeatedly from 3–96 hpf; hatching assessed at 48/72/96 hpf.	3, 6, 10, 24, and 96 hpf	Early morphology (3–95 hpf): no significant morphological changes at 6, 10, and 24 hpf across groups. Survival (96 hpf): survival rate significantly decreased in mg/L PHA and 1 mg/L PBAT, and in 100 mg/L PGA, PLA, PBS, and PHA groups. Hatching: at 48 hpf, hatching rates were significantly increased in high-concentration MP groups; at 72 and 96 hpf, hatching showed a decreased trend. Larval morphometrics (96 hpf): no significant malformations at 96 hpf, but body length and head area were markedly reduced in all exposure groups except 1 mg/L PGA; eye area decreased except in 1 mg/L PGA and 1 mg/L PBAT. Retinal histology (5 dpf): IPL thickness was significantly reduced in 1 mg/L PGA and 100 mg/L PBAT; ONL thickness significantly decreased in 1 mg/L PBS and 1 mg/L PBAT; RGL thickness significantly decreased in all treatment groups. Eye/retina gene expression (5 dpf): 100 mg/L PGA, PLA, and PBAT significantly decreased pax6a, pax6b, rx1, gnat2, grk1b, and opn1mw1; 100 mg/L PBS increased pax6b, gnat2, grk1b, and opn1mw1 but reduced rx1; 100 mg/L PHA increased pax6a, gnat2, and grk1b but reduced rx1.	[62]
	PVC/mean size ~250 µm/100, 200, 300, 400 ppm	Phenotype endpoints: survival, hatching, edema, tail malformation.	30 days	Survival: no mortality reported through 120 h post-fertilization (hpf) across all exposure scenarios (MP-only, phenanthrene-only, co-exposure). Hatching: hatching rate was reported as not affected, including under the study’s “extremely high” exposure conditions. Morphology/teratogenic endpoints (monitored at 48, 72, 96, 120 hpf): edema rate and tail malformation rate were recorded as developmental indicators. Edema: significant edema increase occurred in the MP-only highest concentration group (A4; 400 ppm) at 120 hpf; phenanthrene-only groups showed no significant edema effect vs. control. Co-exposure edema: increased edema was observed at lower MP concentration (200 ppm) in co-exposure groups AB3 and AB4, interpreted in the paper as a synergistic toxicological effect because phenanthrene alone did not produce edema. Tail malformation: MP-only exposure produced a significantly higher tail malformation rate at high MP concentrations (300 and 400 ppm) at 120 hpf (reported up to 19.1%). Phenanthrene-only tail malformation: significant differences vs. control was observed at 72, 96, and 120 hpf, but the paper states the degree was smaller than in MP-only high-dose effects. Co-exposure tail malformation: a slight increase was noted at 48 and 72 hpf, which was reported to disappear with longer exposure.	[54]
	PET/irregular fragments/Average 30–100 µm/1 mg/L & 10 mg/L	Growth (length/weight)	0 to 96- or 120-h post-fertilization (hpf)	Significant growth alterations.	[55]
	Virgin & weathered PS & PE/spherical/virgin 10 µm and 30 µm; weathered PS (lab prepared, ~1–7 µm range) and weathered PE (lab prepared, ~1–10 µm range)/virgin MPs targeted ~105–106 particles/L, Weathered MPs targeted ~104 particles/L	Survival/mortality, teratogenic outcomes (spinal/tail defects, edema)	10 days	Virgin PS: 10–20% (PS 30 µm ~10%; PS 10 µm ~20%; not significant vs. control), weathered MPs: ~80–82% (weathered PS ~80%; weathered PE ~82%; highly significant). Malformations: weathered groups: spinal malformations ~13–14%, bent tails ~21%, significantly higher than control, virgin PS groups: low/non-significant malformations.	[39]
	PS/microsphere/5 μm/1 mg/L	Mortality/survival and body length	2 h post-fertilization (hpf) and continued to 7 days post-fertilization (dpf).	Embryo hatching: no significant effect on hatching. Heart rate: no significant effect on heart rate. Mortality: increased embryo and larval cumulative mortality during the 7-day exposure Growth (body length): no significant effect on growth.	[45]
Larvae	PS & PVC/spherical/PS (spherical ~7.0 µm mean size) PVC (spherical ~3.8 µm mean size)/both MPs 20 mg L−1	Survival and development: daily mortality, morphological abnormalities	10 days	PS MPs alone: higher mortality than PVC (10–20%), strong locomotion suppression. PVC MPs alone: moderate mortality, moderate suppression.	[57]
	PS/virgin & photo-aged/spherical beads/1 µm/0, 1, 10, and 100 µg/L both virgin & photo-aged	Growth and development: body length and survival rate	96 h post-fertilization (hpf)	More strongly inhibited larval growth, and ↓ survival rate in photo-aged PS.	[63]
	PS/microbeads/5 ± 3 μm & 50 ± 3 μm/10 mg/L at 5 μm (MPs-5) or 50 μm (MPs-50)	Growth indices: body weight, body length, condition factor (K = W/L3 × 100)	Up to 96 hpf	Developmental endpoints: PS-MPs 10 mg/L (5 μm): malformation 31.3%; mortality 11.1%; hatching 72 hpf 72.2%, 96 hpf 87.5%; PS-MPs 10 mg/L (50 μm): malformation 27.8%; mortality 8.33%; hatching 72 hpf 68.1%, 96 hpf 88.9%.	[56]
	Virgin & UV-aged PA/irregular fragments/~5 µm (mean particle diameter)/0, 10, 100, and 1000 µg/L	Growth and development: body length and survival rate	2 days post-fertilization (dpf) → 10 dpf	10 (µg/L): Slight growth inhibition (photo-aged > pristine). 100 (µg/L): reduced body length; intestinal structural changes. 1000 (µg/L): significant growth inhibition; severe intestinal damage; impaired lipid adsorption; photo-aging significantly enhanced PA toxicity.	[48]
	Virgin & photo-aged PS/virgin: 10 μm Photo-aged: 6.5 μm/0.1–100 μg/L for both V-PS and P-PS.	Neurotransmission: neurotransmitters (5-HT, GABA, DA, ACh); enzymes (AChE, ChAT, ChE)	1 hfp to 120 hpf	V-PS significantly increased neurotransmitter levels and cholinergic enzyme activity; response trends with concentration and IBR weighting toward neurotransmitter disruption; only V-PS has the effect on the neurotransmitter level of the zebrafish.	[47]
	PS/~25 μm in diameter/25, 250 μg/L	Neurobehavior: light–dark locomotor response (movement distance, max acceleration, average velocity); neurodevelopment genes: gap43, α1-tubulin	Early dpf stages	No significant change in neurobehavioral or neurodevelopment-related gene expression (p > 0.05).	[64]

Abbreviations: PS—polystyrene; PP—polypropylene; PE—polyethylene; PES—polyester; PVC—polyvinyl chloride; PGA—polyglycolic acid; PBAT—polybutylene adipate terephthalate; PLA—polylactic acid; PHA—polyhydroxyalkanoate; PBS—polybutylene succinate; PET—polyethylene terephthalate; PA—polyamide; MPs—microparticles; hpf—hours post-fertilization; dpf—days post-fertilization; DNMT—DNA methyltransferase; PCNA—proliferating cell nuclear antigen; 5-HT—5-hydroxytryptamine; GABA—gamma-aminobutyric acid; DA—dopamine; Ach—acetylcholine; AChE—acetylcholinesterase; ChAT—choline acetyltransferase; ChE—cholinesterase; AgNP—silver nanoparticles; GFP—green fluorescent protein; UV—ultraviolet; PPM—parts per million; ↑—increased; ↓—decreased; →—progression.

Table 1. Developmental/neurodevelopmental effects of pristine/virgin/undisclosed and UV/photo-aged MP exposures.

Life Stage	Plastic Characterization (Type/Shape/Size/Concentrations)	Endpoints	Exposure Time	Effects	Ref.
Adult	PP/Spherical/Virgin: 33.2 ± 14.4 μm/UV: 20.8 ± 10.5 μm/50 mg/L for PP and UV-PP	Survival rate, hatching rate, time to hatch	14 days	Excretion began immediately after MP removal, GI residual after 1 day: PP = 7.9%, UV-PP = 12.3%, elimination half-life: PP = 0.78 days, UV-PP = 0.38 days, 99.9% excreted after 5 days.	[42]
	PE & PES/Fragmented beads/average size of 180 ± 210 µm/1 mg/L both PE & PES	Growth and development: body length and survival rate	96 h (4 days) acute exposure	No significant developmental toxicity.	[43]
	PS/5 µm/2 mg/L	Hatching rate (72 hpf), body length (7 dpf), heart rate, and malformation rate (pericardial edema, yolk sac edema, spinal deformity)	14 days	No recorded mortality for PS MP exposure.	[58]
	PE & PES/Fragments/PE mean size: 180 ± 210 µm PES mean length: 350 ± 220 µm/0.2 mg/L, 1 mg/L both PE & PES	Cardiac and developmental: heart rate, pericardial edema, cardiac looping, blood flow abnormalities, survival and hatching	30 days	No mortality observed, no major difference between 0.2 and 1 mg/L (threshold effect).	[45]
	PS/Spherical beads/5 µm diameter/20 µg/L	Growth indices: body weight, body length, condition factor (K = W/L3 × 100)	21 days	No mortality occurred; final body length and body weight did not change.	[44]
	PGA/~1 μm in diameter/1 mg/L & 100 mg/L	Neurochemistry: brain 5-HT system	28 days	↓ brain 5-HT both 1 mg/L & 100 mg/L. Serotonin-pathway gene expression: ↑ tph1b while ↓ tph1a. Brain inflammatory gene expression: ↑ (il-1β, tnf-α, il-10) after PGA exposure.	[47]
Embryos	PS/Spherical beads/1, 5 µm/10, 100, 1000 µg/L	Survival rate, hatching rate, time to hatch	96 h (4 days post-fertilization)	PS (10 µg/L) No significant effects on survival or hatching. Minor but non-significant increase in pericardial edema (5%). PS (100 µg/L) ↓ Hatching rate (~10% reduction); slight increase in tail curvature (8–10%). PS (1000 µg/L) Significant developmental delay; ↓ hatching (−25%), ↓ heart rate (−13% vs. control), ↑ deformities (22% embryos malformed).	[37]
	Virgin & Photo-aged PS/1 μm diameter/0, 0.1, 1, 10, 100 μg/L for virgin and photo-aged PS MPs	Embryo development and mortality (daily observation); neurotoxicity-focused endpoints: locomotor behavior, neurotransmitter changes, neuronal development and gene expression related to neurotransmission/adipocytokine signaling.	24 h	Motor neuron development (Tg(hb9-GFP), 120 hpf): A-PS exposure reduced motor neurons in brain and spinal cord relative to control; GFP fluorescence intensity decreased from 238 ± 2.58 AU (control) to 234 ± 2.88 (0.1 μg/L), 232 ± 4.97 (1 μg/L), 230 ± 3.95 (10 μg/L), and 229 ± 2.53 AU (100 μg/L) (p < 0.05). Neurodevelopment- and nervous system-related gene expression (120 hpf): A-PS significantly altered expression of nervous system function/development genes (nestin, gfap, manf, shha, alpha–tubulin, mbp). Relative to control, A-PS significantly inhibited gabra1 and manf, and significantly elevated nestin, gfap, alpha–tubulin, and mbp.	[48]
	Virgin & Artificially weathered PP & PS/PS: ~15–36 µm PP: ~50–148 µm; after weathering: (≤230 µm)/Environmentally relevant (particle based): 2000, 20,000, 200,000 MP·L−1; high concentrations (mass based): 12.5, 25, 50, 100 mg·L−1	Mortality, hatching rate	96 h (outcomes recorded at 24/48/72/96 h; heart rate at 72 h; length at 96 h)	2000 MP·L−1: ↓ heart rate, ↓ body length. 20,000 MP·L−1: sublethal growth effects. 200,000 MP·L−1: no linear increase in toxicity. 12.5–100 mg·L−1: no significant embryotoxicity.	[38]
	PS/spherical/0.1 μm diameter/0, 0.1, 1, 10, 50, 100 mg/L	Development: cumulative mortality, hatching, malformation (48–96 hpf); morphometrics: yolk sac area, pericardial edema, body length, eye size, heart rate.	96 h	PS attenuated AgNP toxicity rather than acting as a strong developmental toxicant at the selected dose.	[49]
Embryos → Larvae	Fluorescent plastic microspheres/spherical/1–5 μm/2 mg/L (~1.09 × 108 particles/L)	Developmental and biodistribution: mortality, growth, distribution of MPs in tissues	2 hpf to 14 dpf	Increased PCNA-positive cells in retina → increased cell proliferation, downregulation of neurogenesis genes (sox2, neuroD, olig2), altered DNMT expression (epigenetic modulation), presence of plastic particles in retina.	[50]
	Not disclosed/MPs: ~1 µm diameter/MPs suspended at 0.006%, 0.0045%, 0.003%, 0.0015% solids	Mortality rate, tail and vascular morphology (larval stage), angiogenesis, growth metric (caudal body length), caudal vein morphometrics, and heart morphology	Exposed for 3 days (1 dpf → 4 dpf)	Developmental toxicity included: pathological changes of caudal vein plexus (angiogenesis abnormalities), caudal tissue impairment and reduced growth/body length, peripheral microcirculation dysfunction (caudal region). Mortality (1 dpf → +1day exposure): mortality from 29.2% (MP1) to 95.8% (MP4), with intermediate values 33.3% (MP2) and 58.3% (MP3). Heart morphology: described as largely transformed (malformed) relative to control zebrafish.	[60]
	PS/Fragmented/2 µm/10 mg/L	Developmental toxicity: blood disorder, heartbeat, hatch/death rates, malformations, morphometrics; neurodevelopmental marker: atoh1a expression in cerebellar area (fluorescent reporter)	24–72 hpf	Developmental toxicity: single PS 157 μm showed no observed effect. Neurodevelopmental toxicity: μ-PS did not change cerebellar fluorescence.	[61]
	PGA, PLA, PBS, PHA, PBAT/No data/1 mg/L and 100 mg/L for each polymer	Survival: assessed repeatedly from 3–96 hpf; hatching assessed at 48/72/96 hpf.	3, 6, 10, 24, and 96 hpf	Early morphology (3–95 hpf): no significant morphological changes at 6, 10, and 24 hpf across groups. Survival (96 hpf): survival rate significantly decreased in mg/L PHA and 1 mg/L PBAT, and in 100 mg/L PGA, PLA, PBS, and PHA groups. Hatching: at 48 hpf, hatching rates were significantly increased in high-concentration MP groups; at 72 and 96 hpf, hatching showed a decreased trend. Larval morphometrics (96 hpf): no significant malformations at 96 hpf, but body length and head area were markedly reduced in all exposure groups except 1 mg/L PGA; eye area decreased except in 1 mg/L PGA and 1 mg/L PBAT. Retinal histology (5 dpf): IPL thickness was significantly reduced in 1 mg/L PGA and 100 mg/L PBAT; ONL thickness significantly decreased in 1 mg/L PBS and 1 mg/L PBAT; RGL thickness significantly decreased in all treatment groups. Eye/retina gene expression (5 dpf): 100 mg/L PGA, PLA, and PBAT significantly decreased pax6a, pax6b, rx1, gnat2, grk1b, and opn1mw1; 100 mg/L PBS increased pax6b, gnat2, grk1b, and opn1mw1 but reduced rx1; 100 mg/L PHA increased pax6a, gnat2, and grk1b but reduced rx1.	[62]
	PVC/mean size ~250 µm/100, 200, 300, 400 ppm	Phenotype endpoints: survival, hatching, edema, tail malformation.	30 days	Survival: no mortality reported through 120 h post-fertilization (hpf) across all exposure scenarios (MP-only, phenanthrene-only, co-exposure). Hatching: hatching rate was reported as not affected, including under the study’s “extremely high” exposure conditions. Morphology/teratogenic endpoints (monitored at 48, 72, 96, 120 hpf): edema rate and tail malformation rate were recorded as developmental indicators. Edema: significant edema increase occurred in the MP-only highest concentration group (A4; 400 ppm) at 120 hpf; phenanthrene-only groups showed no significant edema effect vs. control. Co-exposure edema: increased edema was observed at lower MP concentration (200 ppm) in co-exposure groups AB3 and AB4, interpreted in the paper as a synergistic toxicological effect because phenanthrene alone did not produce edema. Tail malformation: MP-only exposure produced a significantly higher tail malformation rate at high MP concentrations (300 and 400 ppm) at 120 hpf (reported up to 19.1%). Phenanthrene-only tail malformation: significant differences vs. control was observed at 72, 96, and 120 hpf, but the paper states the degree was smaller than in MP-only high-dose effects. Co-exposure tail malformation: a slight increase was noted at 48 and 72 hpf, which was reported to disappear with longer exposure.	[54]
	PET/irregular fragments/Average 30–100 µm/1 mg/L & 10 mg/L	Growth (length/weight)	0 to 96- or 120-h post-fertilization (hpf)	Significant growth alterations.	[55]
	Virgin & weathered PS & PE/spherical/virgin 10 µm and 30 µm; weathered PS (lab prepared, ~1–7 µm range) and weathered PE (lab prepared, ~1–10 µm range)/virgin MPs targeted ~105–106 particles/L, Weathered MPs targeted ~104 particles/L	Survival/mortality, teratogenic outcomes (spinal/tail defects, edema)	10 days	Virgin PS: 10–20% (PS 30 µm ~10%; PS 10 µm ~20%; not significant vs. control), weathered MPs: ~80–82% (weathered PS ~80%; weathered PE ~82%; highly significant). Malformations: weathered groups: spinal malformations ~13–14%, bent tails ~21%, significantly higher than control, virgin PS groups: low/non-significant malformations.	[39]
	PS/microsphere/5 μm/1 mg/L	Mortality/survival and body length	2 h post-fertilization (hpf) and continued to 7 days post-fertilization (dpf).	Embryo hatching: no significant effect on hatching. Heart rate: no significant effect on heart rate. Mortality: increased embryo and larval cumulative mortality during the 7-day exposure Growth (body length): no significant effect on growth.	[45]
Larvae	PS & PVC/spherical/PS (spherical ~7.0 µm mean size) PVC (spherical ~3.8 µm mean size)/both MPs 20 mg L−1	Survival and development: daily mortality, morphological abnormalities	10 days	PS MPs alone: higher mortality than PVC (10–20%), strong locomotion suppression. PVC MPs alone: moderate mortality, moderate suppression.	[57]
	PS/virgin & photo-aged/spherical beads/1 µm/0, 1, 10, and 100 µg/L both virgin & photo-aged	Growth and development: body length and survival rate	96 h post-fertilization (hpf)	More strongly inhibited larval growth, and ↓ survival rate in photo-aged PS.	[63]
	PS/microbeads/5 ± 3 μm & 50 ± 3 μm/10 mg/L at 5 μm (MPs-5) or 50 μm (MPs-50)	Growth indices: body weight, body length, condition factor (K = W/L3 × 100)	Up to 96 hpf	Developmental endpoints: PS-MPs 10 mg/L (5 μm): malformation 31.3%; mortality 11.1%; hatching 72 hpf 72.2%, 96 hpf 87.5%; PS-MPs 10 mg/L (50 μm): malformation 27.8%; mortality 8.33%; hatching 72 hpf 68.1%, 96 hpf 88.9%.	[56]
	Virgin & UV-aged PA/irregular fragments/~5 µm (mean particle diameter)/0, 10, 100, and 1000 µg/L	Growth and development: body length and survival rate	2 days post-fertilization (dpf) → 10 dpf	10 (µg/L): Slight growth inhibition (photo-aged > pristine). 100 (µg/L): reduced body length; intestinal structural changes. 1000 (µg/L): significant growth inhibition; severe intestinal damage; impaired lipid adsorption; photo-aging significantly enhanced PA toxicity.	[48]
	Virgin & photo-aged PS/virgin: 10 μm Photo-aged: 6.5 μm/0.1–100 μg/L for both V-PS and P-PS.	Neurotransmission: neurotransmitters (5-HT, GABA, DA, ACh); enzymes (AChE, ChAT, ChE)	1 hfp to 120 hpf	V-PS significantly increased neurotransmitter levels and cholinergic enzyme activity; response trends with concentration and IBR weighting toward neurotransmitter disruption; only V-PS has the effect on the neurotransmitter level of the zebrafish.	[47]
	PS/~25 μm in diameter/25, 250 μg/L	Neurobehavior: light–dark locomotor response (movement distance, max acceleration, average velocity); neurodevelopment genes: gap43, α1-tubulin	Early dpf stages	No significant change in neurobehavioral or neurodevelopment-related gene expression (p > 0.05).	[64]

Abbreviations: PS—polystyrene; PP—polypropylene; PE—polyethylene; PES—polyester; PVC—polyvinyl chloride; PGA—polyglycolic acid; PBAT—polybutylene adipate terephthalate; PLA—polylactic acid; PHA—polyhydroxyalkanoate; PBS—polybutylene succinate; PET—polyethylene terephthalate; PA—polyamide; MPs—microparticles; hpf—hours post-fertilization; dpf—days post-fertilization; DNMT—DNA methyltransferase; PCNA—proliferating cell nuclear antigen; 5-HT—5-hydroxytryptamine; GABA—gamma-aminobutyric acid; DA—dopamine; Ach—acetylcholine; AChE—acetylcholinesterase; ChAT—choline acetyltransferase; ChE—cholinesterase; AgNP—silver nanoparticles; GFP—green fluorescent protein; UV—ultraviolet; PPM—parts per million; ↑—increased; ↓—decreased; →—progression.

4. Physiological and Oxidative Response of Zebrafish Exposed to Microplastics

Across life stages, oxidative stress emerged as the most consistent physiological response of zebrafish to MP exposure. For example, PE MPs with an average diameter of 40 ± 10 µm at 100 µg/L for 21 days resulted in increased superoxide dismutase (SOD) and catalase (CAT) activities, accompanied by mild intestinal villi damage, suggesting activation of antioxidant defense mechanisms in response to oxidative stress (Figure 9). In another study using 1–5 µm spherical microplastics at 2 mg/L, zebrafish larvae exhibited significant increases in ROS production, lipid peroxidation (LPO), and alterations in multiple antioxidant biomarkers including SOD, CAT, GPx, GST, GR, and GSH/GSSG, together with inhibition of acetylcholinesterase activity and reduced physiological capacity [65]. Chronic exposure to weathered polyethylene microplastics at 1 µg/L for 40 days caused significant modulation of immune and oxidative stress responses, including elevated plasma cortisol levels, increased antimicrobial and lysozyme activity, and changes in hematological parameters, indicating that oxidative stress responses were associated with systemic physiological stress during prolonged exposure (Table 2) [66].

Figure 9. (a) Principal component analysis (PCA) illustrating the differential expression of oxidative stress-, antioxidant-, neurotoxicity-, and apoptosis-related genes following MP exposure. The blue vectors represent individual genes, with arrow length indicating their contribution to sample separation and direction indicating their association with the different exposure groups (Cu, C, MPs, and Mix). Genes involved in oxidative stress (e.g., sod, cat, gclc, gstp1), apoptosis (casp3, casp8, casp9), neurotoxicity (ache, slc6a3, slc6a4a, slc6a4b, tph1a, tph2), and cellular proliferation (pcna) were differentially regulated following exposure to MPs and their mixtures with co-contaminants [65]. (b) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway heatmap showing alterations in biological pathways following exposure to virgin polyethylene (VPE) and aged polyethylene (APE) MPs relative to the control. Red and blue colors indicate pathway activation and suppression, respectively, while asterisks (*) denote statistically significant pathway enrichment. Significant alterations were observed in pathways associated with organismal systems, cellular processes, signal transduction, xenobiotic metabolism, amino acid metabolism, lipid metabolism, endocrine function, immune responses, and digestive system regulation, indicating extensive metabolic and cellular dysfunction induced by microplastic exposure [67]. (c) Relative expression and biochemical activities of oxidative stress and intestinal barrier biomarkers, including SOD, IL-1β, CLDN5, OCLN, ZO-1, and D-Lac, across the Control, solvent control (S.C.), PP, and UV-PP treatment groups. Different lowercase letters above the bars indicate statistically significant differences among treatments (p < 0.05), whereas error bars represent experimental variability. The observed alterations demonstrate enhanced oxidative stress, inflammatory responses, and disruption of intestinal barrier integrity following exposure to virgin and UV-weathered polypropylene MPs [42]. (d) Integrated schematic summarizing the molecular and physiological mechanisms underlying MP toxicity in zebrafish larvae. Differentially expressed antioxidant, inflammation, apoptosis, and stress-related genes are represented by colored circles and directional triangles, where red and blue symbols indicate increased and decreased expression, respectively. The chord diagram illustrates the interactions among oxidative stress, apoptosis, inflammation, xenobiotic metabolism, immune dysfunction, motor impairment, and survival. The schematic further demonstrates that microplastic exposure promotes reactive oxygen species (ROS) generation, oxidative stress, inflammatory responses, apoptosis, dysplasia, behavioral abnormalities, histopathological alterations, and increased mortality compared with the control group, highlighting the complex molecular pathways involved in MP-induced toxicity [68].

Figure 9. (a) Principal component analysis (PCA) illustrating the differential expression of oxidative stress-, antioxidant-, neurotoxicity-, and apoptosis-related genes following MP exposure. The blue vectors represent individual genes, with arrow length indicating their contribution to sample separation and direction indicating their association with the different exposure groups (Cu, C, MPs, and Mix). Genes involved in oxidative stress (e.g., sod, cat, gclc, gstp1), apoptosis (casp3, casp8, casp9), neurotoxicity (ache, slc6a3, slc6a4a, slc6a4b, tph1a, tph2), and cellular proliferation (pcna) were differentially regulated following exposure to MPs and their mixtures with co-contaminants [65]. (b) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway heatmap showing alterations in biological pathways following exposure to virgin polyethylene (VPE) and aged polyethylene (APE) MPs relative to the control. Red and blue colors indicate pathway activation and suppression, respectively, while asterisks (*) denote statistically significant pathway enrichment. Significant alterations were observed in pathways associated with organismal systems, cellular processes, signal transduction, xenobiotic metabolism, amino acid metabolism, lipid metabolism, endocrine function, immune responses, and digestive system regulation, indicating extensive metabolic and cellular dysfunction induced by microplastic exposure [67]. (c) Relative expression and biochemical activities of oxidative stress and intestinal barrier biomarkers, including SOD, IL-1β, CLDN5, OCLN, ZO-1, and D-Lac, across the Control, solvent control (S.C.), PP, and UV-PP treatment groups. Different lowercase letters above the bars indicate statistically significant differences among treatments (p < 0.05), whereas error bars represent experimental variability. The observed alterations demonstrate enhanced oxidative stress, inflammatory responses, and disruption of intestinal barrier integrity following exposure to virgin and UV-weathered polypropylene MPs [42]. (d) Integrated schematic summarizing the molecular and physiological mechanisms underlying MP toxicity in zebrafish larvae. Differentially expressed antioxidant, inflammation, apoptosis, and stress-related genes are represented by colored circles and directional triangles, where red and blue symbols indicate increased and decreased expression, respectively. The chord diagram illustrates the interactions among oxidative stress, apoptosis, inflammation, xenobiotic metabolism, immune dysfunction, motor impairment, and survival. The schematic further demonstrates that microplastic exposure promotes reactive oxygen species (ROS) generation, oxidative stress, inflammatory responses, apoptosis, dysplasia, behavioral abnormalities, histopathological alterations, and increased mortality compared with the control group, highlighting the complex molecular pathways involved in MP-induced toxicity [68].

Lower concentrations of MPs often induced compensatory antioxidant responses, whereas higher concentrations produced sustained oxidative imbalance and activation of cellular damage pathways. For example, exposure of zebrafish to PGA MPs at 1 mg/L and 100 mg/L for 28 days resulted in significant physiological alterations associated with oxidative stress. At 1 mg/L, early antioxidant responses and intestinal barrier disturbances were observed, including dysregulation of gut-related signaling pathways and microbial imbalance. At 100 mg/L, stronger systemic responses occurred, including enhanced inflammatory signaling and neurochemical alterations linked to oxidative stress pathways [69]. Similarly, experiments using PS microbeads (5 ± 3 µm and 50 ± 3 µm) at 10 mg/L in zebrafish embryos demonstrated strong oxidative stress responses. Exposure increased ROS production, elevated lipid peroxidation markers such as malondialdehyde (MDA), and activated antioxidant enzymes including SOD and CAT. In addition, oxidative damage was associated with DNA oxidative damage markers (8-OHdG) and altered expression of apoptosis-related genes, including upregulation of p53, Bax, and caspase-3/8/9 and downregulation of the anti-apoptotic gene Bcl-2, indicating activation of apoptosis pathways following oxidative stress exposure [56].

Intestinal toxicity and barrier dysfunction were recurrent findings. Histopathological changes included villus shortening, epithelial shedding, vacuolization, goblet cell reduction, and tight-junction gene downregulation (cldn5, zo-1), accompanied by increased D-lactate levels indicating compromised barrier integrity (Table 2) [48]. Zebrafish exposed to PP and UV-weathered PP MPs (33.2 ± 14.4 µm for virgin PP and 20.8 ± 10.5 µm for UV-PP) at 50 mg/L for 14 days resulted in clear intestinal mucosal damage, characterized by villus structural disruption, vacuolization, ciliary defects, and a 34% decrease in goblet cells in PP treatments and 51% reduction in UV-PP groups [42]. Inflammatory responses were also evident following MP exposure. In the same experiment, expression of inflammatory cytokines was significantly elevated, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), indicating activation of intestinal immune responses associated with tissue injury and oxidative stress [42]. Similarly, exposure to fragmented PE and PES microplastics (180 ± 210 µm and 350 ± 220 µm) at concentrations of 0.2 and 1 mg/L for 30 days resulted in metabolic disturbances in intestinal tissues and disruption of lipid metabolism pathways, suggesting that microplastics interfere with normal intestinal physiological processes [46].

Gut microbiota profiling further confirmed significant dysbiosis after microplastic exposure. Studies reported increases in Proteobacteria abundance and shifts in Fusobacteria and Firmicutes populations, indicating disruption of microbial community structure. In addition, metabolomic analyses revealed disturbances in key metabolic pathways, including purine metabolism, bile acid biosynthesis, arginine metabolism, and lipid metabolism, demonstrating that intestinal microbial imbalance can influence host metabolic regulation (Table 2) [68,70]. Also, chronic exposure of zebrafish to PS MPs (5 µm) at 20 µg/L for 21 days resulted in significant alterations in hepatic metabolic parameters. Specifically, hepatic pyruvate, triglycerides (TG), and total cholesterol (T-CHO) levels were significantly reduced, indicating disruption of lipid and carbohydrate metabolism [44].

Although several glycolipid metabolism genes (e.g., gk, hk1, pepckc, aco, cpt1, ppar-α, acc, and fas) did not show significant transcriptional changes, oxidative stress markers were affected, including increased catalase (CAT) activity and reduced glutathione (GSH) levels, together with suppression of oxidative stress-related genes such as Mn-sod, Nrf2, Keap1, Gpx, and Bcl2, demonstrating impaired antioxidant regulatory pathways in hepatic tissue (Table 2) [71,72]. Histopathological studies also demonstrated liver tissue damage associated with MP exposure. Exposure to PS MPs of varying particle sizes (10 µm, 40 µm, and 200 µm) at 100 µg/L for 30 days resulted in hepatocyte vacuolization, nuclear abnormalities, and structural alterations of liver tissue, indicating hepatic injury. Oxidative stress biomarkers further showed size-dependent responses, where superoxide dismutase (SOD) activity increased as the particle size decreased, while CAT activity significantly decreased in groups exposed to the smallest particles, demonstrating that smaller microplastics induced stronger oxidative stress responses in liver tissue [73].

Metabolomic analyses further revealed systemic metabolic disturbances in zebrafish exposed to MPs. In the study of Dimitriadi et al. [74], zebrafish were exposed to PS MPs with a particle size of approximately 5 µm at concentrations of 1, 10, and 100 mg/L, and metabolomic profiling of cardiac tissues revealed substantial alterations in central energy metabolism pathways. Several intermediates of the tricarboxylic acid (TCA) cycle, including succinic acid (~75%) and α-ketoglutaric acid (~56%), were significantly reduced, while pyruvic acid increased by approximately 38% relative to the control group. These changes indicate disruption of mitochondrial metabolic processes and oxidative phosphorylation, suggesting that MP exposure interferes with cellular energy production and metabolic regulation.

Furthermore, endocrine and reproductive endpoints demonstrate interference with hormonal regulation following MP exposure. For example, exposure of adult zebrafish to PS MPs with a particle size of ~100 µm at a concentration of 40.1 µg/L for 21 days resulted in significant endocrine disruption [75]. This exposure increased ovarian testosterone levels by approximately 75% and brain testosterone levels by about 39.3% compared with controls. Histological examination of ovarian tissue revealed oocyte maturation arrest characterized by an increased proportion of immature stage I–II follicles and reduced numbers of mature follicles, indicating impaired reproductive function and chronic anovulation-like conditions. In addition, oxidative stress biomarkers such as malondialdehyde (MDA) increased to approximately 0.015 µM/mg ovarian tissue, suggesting oxidative damage associated with endocrine disruption [76].

Moreover, chronic exposure to PE MPs with a particle size of approximately 25 µm at 100 µg/L for 35 days significantly altered endocrine regulation in zebrafish. Hormonal assays showed disruption of reproductive hormones including estradiol (E2), testosterone (T), and 11-keto testosterone (11-KT), accompanied by altered expression of genes within the hypothalamic–pituitary–gonadal–liver (HPGL) axis such as gnrhr2, gnrhr3, cyp11a, cyp19a, erα, and vtg, which are essential regulators of steroidogenesis and reproductive development. Histological analysis also revealed pathological ovarian changes including loss of contact between the oocyte membrane and follicular cell layers and cellular degeneration, confirming reproductive toxicity following PE exposure [77]. Additionally, endocrine disruption involving the thyroid axis has been reported following exposure to PS MPs with a particle size of approximately 2 µm at concentrations of 0.1 mg/L and 1 mg/L over 63 days [78].

These exposures enhanced maternal transfer of thyroid hormones (T3 and T4) to offspring while simultaneously reducing thyroid hormone levels in the F2 generation, demonstrating that microplastics can interfere with endocrine signaling across generations [78]. Artificially aged PLA MPs with an approximate particle size of ~100 µm at a concentration of 5 mg/L also induced reproductive toxicity during 5 weeks of exposure [78]. In this study, aged PLA particles caused ovarian structural damage and disrupted steroid hormone balance, and these maternal exposures produced offspring with increased mortality, reduced hatching rates, and decreased body length, indicating that weathered bioplastic particles can exert transgenerational developmental effects [78].

Additionally, neurophysiological biomarkers and cardiovascular endpoints further demonstrate systemic involvement following microplastic exposure. Neurotoxicity was frequently assessed through acetylcholinesterase (AChE) activity, which was significantly altered. For instance, zebrafish exposed to an undisclosed type of MPs (~1–5 µm) at a concentration of 2 mg/L from 2 hpf to 14 dpf exhibited significant inhibition of AChE activity, accompanied by elevated oxidative stress biomarkers including ROS, lipid peroxidation, and changes in antioxidant enzymes (SOD, CAT, GST, and GR), linking oxidative stress responses with neurophysiological dysfunction [50]. Similarly, exposure of zebrafish embryos and larvae to PS MPs of 1 µm diameter at concentrations of 0.1, 1, and 10 mg/L for 21 days induced oxidative stress responses characterized by increased MDA levels and altered antioxidant enzyme activity, which were associated with reduced swimming performance and behavioral changes indicative of neurotoxicity [79].

Cardiovascular effects of MPs have also been widely reported. Exposure of zebrafish embryos to PS MPs (1–5 µm) at concentrations of 10, 100, and 1000 µg/L resulted in significant reductions in heart rate at higher concentrations, demonstrating cardiotoxic responses during early developmental stages [37]. In addition, experiments assessing PS MPs at concentrations of 1, 10, and 100 µg/L under co-exposure with microcystin-LR showed thrombus formation in the caudal vein, inhibition of angiogenesis in vascular tissues, and increased oxidative stress and inflammatory gene expression, indicating disruption of cardiovascular development pathways [63]. Further metabolomic and histopathological studies using 5 µm PS MPs at concentrations of 1, 10, and 100 mg/L demonstrated cardiac oxidative damage characterized by a 528.5% increase in lipid peroxidation and approximately 100-fold increase in DNA damage markers, together with activation of apoptosis pathways including increased Bax/Bcl-2 ratio and elevated caspase-3 and caspase-9 expression, indicating severe cardiac injury following high-dose exposure [73].

Lastly, bioaccumulation and depuration dynamics demonstrated rapid gastrointestinal uptake and variable elimination kinetics in zebrafish exposed to MPs. For example, zebrafish exposed to PP MPs with an average particle size of 33.2 ± 14.4 µm (virgin PP) and 20.8 ± 10.5 µm (UV-weathered PP) at a concentration of 50 mg/L for 14 days showed rapid ingestion and accumulation within the gastrointestinal tract. Following removal of MPs from the exposure medium, depuration occurred relatively quickly, with residual particles remaining at 7.9% for virgin PP and 12.3% for UV-weathered PP after 1 day, and approximately 99.9% of particles eliminated within 5 days. The elimination half-life was 0.78 days for virgin PP and 0.38 days for weathered PP, indicating faster depuration but greater short-term retention of weathered particles due to stronger biological interaction [42].

Although studies examining PS MPs (~5 µm) at concentrations of 1, 10, and 100 mg/L reported widespread tissue distribution following ingestion, with particles detected in the gastrointestinal tract, liver, and other internal tissues, suggesting systemic bioavailability of smaller particles after intestinal uptake [73]. Sex-specific responses were also observed during chronic exposure experiments. In one study, zebrafish exposed to PS MPs (~5 µm) at 20 µg/L for 21 days exhibited significant alterations in gut microbiota composition and metabolic responses. Female zebrafish displayed stronger microbiota dysbiosis, including greater shifts in dominant microbial taxa and associated metabolic pathways, which coincided with reproductive impairment and altered lipid metabolism, indicating that sex-dependent physiological differences can influence the biological impact of microplastic accumulation [71].

Table 2. Physiological effects of pristine/virgin/unspecifies and UV/photo-aging in MP exposure.

Life Stage	Plastic Characterization (Type/Shape/Size/Concentrations)	Endpoints	Exposure Time	Effects	Ref.
Adult	PE/Spherical/Average diameter of 40 ± 10 µm/100 µg/L	Oxidative stress biomarkers: SOD, CAT, GPx, MDA, T-AOC.	21 d	100 µg/L: Slight oxidative stress (↑ SOD, CAT activities); minor intestinal villi damage.	[80]
	PS/Spherical beads/1 μm & 3 μm/0.01, 0.1, 1.0, 10.0 mg/L	Cardiac physiology (heart rate at 72 h); Redox homeostasis/oxidative stress biomarkers.	96 h (4 days post-fertilization)	↑ in heart rate both concentrations with more strongly at 10 mg/L. ↓ ROS content at 10 mg/L; ↑ Lipid hydroperoxides but more significant in 10 mg/L. Antioxidant enzymes: GPX: Unaffected; GR: ↑ significantly only in 1 mg/L; SOD: ↑ significantly only in 10 mg/L.	[37]
	Virgin & UV PP/Virgin: 33.2 ± 14.4 μm UV: 20.8 ± 10.5 μm/50 mg/L both V & UV	Histopathology: Intestinal villi length, mucus secretion, goblet cell number, vacuolization, ciliary defects. Gene expression: Sod1, IL-1β, CLDN5, Oclna, ZO-1. Enzyme biomarkers: SOD, IL-1β (inflammation), and D-lactate (intestinal permeability). Gut microbiome: Alpha diversity (Chao1, Shannon), Beta diversity (PCoA, UniFrac), OTU abundance, taxonomic shifts at phylum & genus levels, KEGG pathway analysis using PICRUSt & MinPath.	14 d	Tissue distribution and accumulation: Both V & UV weathered PP particles were detected in the GI tract, liver, and gills. Peak GI burden (day 3): PP = 383.4 ± 50.3 particles; UV-PP = 2053.7 ± 371.4 particles. Excretion kinetics (GI): Elimination half-life PP = 0.78 d; UV-PP = 0.38 d; after 5 days of depuration, both MPs reached 99.9% excretion. Intestinal histopathology: Exposure to PP and UV-PP produced mucosal damage characterized by structural damage, vacuolization, ciliary defects, mucus secretion, and reduced goblet cells. Goblet cells: Decreased by 34% (PP) and 51% (UV-PP). Oxidative stress/inflammation markers (gut): SOD mRNA upregulated significantly in UV-PP; il-1β mRNA upregulated significantly in both PP and UV-PP vs. S.C; SOD and IL-1β enzyme-level changes were consistent with gene expression patterns. Tight junction/barrier markers: cldn5 and zo-1 transcription downregulated in PP and UV-PP; D-lactate (D-Lac) increased. Gut microbiota diversity and composition: Shannon index increased significantly in the PP group; PCoA indicated microbiome shifts after PP and UV-PP exposure. Differential taxa (reported comparisons): Rhizobium, Gemmobacter, and Cloacibacterium were significantly higher in PP than UV-PP; Luteolibacter and Rodobacter were significantly higher in UV-PP; Porphyromonadaceae and Aeromonas tended to decrease in MP-exposed groups.	[41]
	Virgin MPs (propriety polymer)/Spherical/1–5 μm/2 mg/L (~1.09 × 108 particles/L)	Biochemical biomarkers: ROS, LPO, SOD, CAT, GPx, GST, GR, GSH/GSSG, LDH, AChE, and MT. Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.), and intestinal barrier genes (zo-1, claudin).	2 hpf to 14 dpf	Significant inhibition of acetylcholinesterase (AChE) activity compared with control, indicating neurotoxicity. AChE inhibition showed correlation with mortality increase and reduced growth. Other biomarkers such as ROS, LPO, SOD, CAT, GST, GR, GSH/GSSG, LDH, significantly affect the overall physiological capacity of the zebrafish.	[65]
	PE & PES/Fragmented beads/Average size of 180 ± 210 µm/1 mg/L both PE & PES	Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.) and intestinal barrier genes (zo-1, claudin). Oxidative stress: ROS production, antioxidant enzymes (SOD, CAT), lipid peroxidation (MDA).	96 h (4 days) acute exposure	PE exposure: Significantly altered lipid metabolism in the intestine and liver, and perturbation of lipid metabolism, fatty acid metabolism, vitamin metabolism, TCA cycle, and amino acid metabolism. PES exposure: Upregulated metabolites included phosphocholine and 2-lysophosphatidylcholine; and downregulation metabolites included triglyceride, 13-HDoHE, n-triacontanol, and phosphatidylserine.	[43]
	Propriety polymer (composition undisclosed/Spherical/1–5 µm/2 mg/L	Molecular responses: Oxidative stress-related genes, detoxification-related genes. Metabolomics: Whole-body metabolomics, LC-MS/MS (QTOF) untargeted metabolomics.	30 d	MPs (2 mg/L): ↑ Metallothionein (MT), ↑ tph1a (serotonin synthesis gene), ↓ LDH (metabolic alteration). Gut microbiota: Increased; Fusobacteria, and Protobacteria; Decreased: Firmicute; Clear dysbiosis in both PE and PES groups.	[64]
	PS/Microbeads/200 µm, 40 µm, 10 µm/100 µg/L	Biochemical biomarkers: ALT, AST (hepatic injury), SOD, CAT, GSH (oxidative stress), integrated biomarker response (IBR).	30 d	MP exposure resulted in liver pathological changes, wherein liver damage included the presence of ballooning of the hepatocytes (vacuolization), nuclear abnormalities (including the formation of pyknotic or peripherally located nuclei), and the presence. CAT activities decreased with the decrease of MPs size, and the smallest MP group showed a significant decrease in CAT under single exposure. In the MP single-exposure groups, SOD activity increased with decreasing plastic size. GSH content significantly rose under the single MP treatments, but the magnitude of GSH increase showed a decline with a decrease in MPs size.	[73]
	PS/5 µm/2 mg/L	Oxidative stress biomarkers: Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), malondialdehyde (MDA). Histopathology: Liver tissue, hematoxylin & eosin (H&E) staining, hepatocyte vacuolization, cellular degeneration, histopathological scoring. Gene expression (qRT-PCR): Metallothionein (mt2), antioxidant-related genes, apoptosis-related genes.	14 d	Gills (Histopathology): There were no histopathological changes in the gills of the fish that were exposed to not contaminated. Intestine (Histopathology): Intestinal changes are minor, including cracking of villi, caused by MPs alone. Liver (histopathology): There was no obvious impact of MPs alone on histopathology.	[44]
	PE & PES/Fragments/PE mean size: 180 ± 210 µm PES mean length: 350 ± 220 µm/0.2 mg/L, 1 mg/L both PE & PES	Whole-body untargeted LC-MS/MS metabolomics to quantify MP-associated shifts in molecular metabolites and enriched metabolic pathways. Gut microbiome physiology and gut microbiota profiling after MP exposure.	30 d	PE-specific metabolomic changes: 0.2 mg/L and 1 mg/L overlapped with minimal separation, while both separated from controls. Both concentrations had no significant different and does not change metabolomics of zebrafish. Gut microbiome (16S metagenomics): Community composition: Dominant phyla included Proteobacteria, Fusobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Verrucomicrobia; Fusobacteria increased (0.3–11.7%) in microplastics-exposed groups vs. control, while Proteobacteria decreased (0.4–9.0%) across exposures vs. control. Potential pathogens were reported only in exposed groups (examples: Mycobacterium in PES2; Aeromonas in PE1/PE2). Diversity: Alpha diversity indices (observed OTU, Shannon, Faith FD, Simpson) were reported as not significantly altered across exposure groups. PE: Physical epithelial damage, oxidative stress, inflammation, disrupted membrane and energy metabolism. PES: Fibers interfered more strongly with gut microbiota interactions, lipid digestion, and endocrine-related metabolism.	[45]
	PVC & PP/Irregular fragments/5–50 µm/PVC MPs (100 µg/L), and PP MPs (100 µg/L)	Intestinal histopathology (H&E); intestinal inflammation biomarkers/gene expressions: TNF-α levels and IL-1β gene expression. Oxidative stress biomarkers/antioxidant-related expression; intestinal metabolomics; and intestinal microbiota	21 d	Intestinal histopathology (H&E): Control intestines showed intact villi shape and regularly arranged epithelial cells. PVC-only and PP-only groups showed slight shedding of intestinal villi. Intestinal inflammation biomarkers/gene expression: TNF-α levels significantly increased in all treatment groups except the cadmium-only group; this includes PVC-only and PP-only. IL-1β gene expression significantly increased in all treatment groups; this includes PVC-only and PP-only. Oxidative stress biomarkers/antioxidant-related expression: No remarkable shifts in SOD activity or MDA levels were reported after exposure to PVC alone or PP alone. PP (but not PVC) significantly increased GPx expression (detoxification-related). Intestinal metabolomics: PVC-only: 32 significant differential metabolites downregulated and 24 upregulated vs. control. PP-only: 85 significant differential metabolites downregulated and 10 upregulated vs. control. KEGG pathway enrichment: both PVC-only and PP-only showed disturbances in purine metabolism and arginine/proline metabolism. PVC-only additionally disrupted primary bile acid biosynthesis and arachidonic acid metabolism. PP-only additionally affected arginine biosynthesis and glutamate metabolism. Intestinal microbiota: At the phylum level, Firmicutes relative abundance increased in PVC-only and PP-only groups. Proteobacteria relative abundance increased in all treatment groups, including PVC-only and PP-only.	[65]
	PS/Spherical beads/2 μm diameter/0.44 mg/L (~108 items/L)	Oxidative biomarkers: SOD, CAT, GSH, and MDA	7 d	Ocular oxidative stress biomarkers (eyes; ELISA; normalized to protein): MP exposure showed no significant main effect on SOD, CAT, GSH, or MDA in the model analysis (p > 0.1). GSH specifically: The MP-only group had significantly higher ocular GSH than the control group. MDA: No significant difference was detected in ocular MDA among groups.	[66]
	PS/Spherical beads/2–4 µm diameter/440 µg/L	Oxidative stress biomarkers: SOD, CAT activities, MDA content, and protein normalization. Histopathology: Villus number, villus height and width, and intestinal wall thickness; and gut microbiota analysis.	21 d	PS beads were observed in the gut at day 1 and day 21 (control showed only autofluorescence). PS distributed along anterior–middle–posterior gut and tended to accumulate in the mid-posterior gut and/or be excreted with fecal pellets. Oxidative stress/anti-oxidant response: CAT activity was significantly increased in the PS group compared with the control group (and AMI group). SOD activity was not reported as significantly increased in the PS group (significance was reported for the PS + AMI group vs. control/PS/AMI). MDA was not reported as significantly increased in the PS group (the significant change reported was a decrease in the AMI group vs. control/PS). Morphometrics: Villus width was significantly increased in the PS-MP group; intestinal wall thickness was significantly decreased in the PS-MP group. The number of villi showed decreasing tendency in the PS-MP group. Gut microbiota: PS-MPs showed an increasing tendency in Proteobacteria, which includes Exiguobacterium, Candidatus paracaedibacter, and Staphylococcus.	[69]
	PS/Spherical beads/5 µm diameter/20 µg/L	Hepatic biochemical parameters: Energy metabolism: glucose, pyruvate, Lipid metabolism: TG, T-CHO, LDL-C, NEFA, oxidative stress: SOD, CAT, GSH, MDA Glycolipid metabolism genes: Glycolysis/gluconeogenesis: Gk, Hk1, Pepckc, β-oxidation: Aco, Cpt1, Ppar-α, Lipid synthesis: Acc, Fas, Ppar-γ, Lipid transport: Apo, Fabp6. Oxidative stress genes: Mn-sod, Cu/Zn-sod, Cat, Nrf2, Keap1, Gpx, Bcl2; Inflammatory genes: IL-1β, IL-6, IL-8, TNF-α, IFN, C3, IL-10.	21 d	Hepatic metabolism: PS: ↓ pyruvate, TG, T-CHO. PS specific significantly changes: Hepatic pyruvate, TG, and T-CHO with significant decreased; PS had no significant effect on NEFA. Hepatic glycolipid metabolism genes: Based from the results, PS had no significant effect on glycolipid metabolism genes. Oxidative biomarkers: CAT activity had a significant increase in PS exposure vs. control.; the GSH content was reduced after 21 days of exposure; oxidative stress-related mRNA such as Mn-sod, Nrf2, Keap1, Gpx, and Bcl2 mRNA levels were significantly inhibited in PS-MPs.	[46]
	Virgin & Photo-aged PLA/~100 µm/5 mg·L−1	Female reproductive endpoints: Gonadosomatic index (GSI), ovary histology (H&E), sex hormones: Testosterone (T), estradiol (E2), Emphasis on E2/T; ovary metabolomics.	5 w	DPLA > UPLA toxicity, ovarian structural damage, disrupted steroid hormones, altered metabolomic pathways, offspring: ↑ mortality, ↓ hatching, ↓ body length.	[48]
	PE/Spherical/146.2 ± 8.9 µm/5 µg/L and 50 µg/L	Oxidative stress & antioxidant biomarkers: Catalase (CAT), glutathione-S-transferase (GST), lipid peroxidation (MDA); ion regulation: Na+/K+-ATPase activity (gills).	10 d & 20 d	5 µg/L: 10 days: ↓ CAT and GST (liver); 20 days: ↑ GST activity (adaptive response), ↑ lipid peroxidation (brain). 50 µg/L: Stronger oxidative stress than 5 µg/L, significant ↑ MDA (brain), marked ↑ Na+/K+-ATPase activity in gills.	[70]
	PS/Spherical beads/1 µm/30 mg/L	MP distribution (gills vs. gut), gut histopathology, oxidative stress enzyme, liver metabolomics, gut microbiota (16S rRNA), sex hormones, and reproductive output.	Chronic (multi-week).	Females accumulated more PS, gut microbiota dysbiosis stronger in females, altered hepatic lipid & energy metabolism, reduced egg production, clear sex-specific toxicity.	[71]
	PS/Fragmented/5 µm/20 & 200 mg/L	Cd accumulation (liver, gut, gill), oxidative stress enzymes, histopathology, inflammatory gene expression.	Chronic (multi-week).	MPs enhanced Cd bioaccumulation, there was an increased trend in oxidative damage and inflammation synergistically increased, MPs acted as toxic vector.	[72]
	PS/Fragmented/5 µm/LMPs: 0.1, 1, 10, 50, 100 mg/L; SMPs: 0.1, 1, 10, 50, 100 mg/L	Antioxidant enzymes: SOD, CAT, GPx, lipid peroxidation (MDA).	96 h post-exposure	SOD activity: At day 4, PS-MP exposure significantly increased hepatic SOD. CAT activity: At day 4, CAT activity was significantly higher and remained significantly higher at day 8. GPx: At day 4, GPx activity was significantly higher and remained significantly higher at day 8. Lipid peroxidation (MDA): PS-MPs had no significant effect on hepatic MDA at day 4 but evidently reduced at day 8.	[49]
	PS/Not disclosed/0, 50, 500 µg/L	Gene expression: qPCR normalization to β-actin, ovarian histology: (oocyte stage scoring; H&E), oxidative stress markers: MDA (lipid peroxidation), SOD, CAT.	60 d	50 and 500 µg/L: Decreased ovarian SOD/CAT activity (oxidative stress), increased NO, increased apoptosis; dose response noted (500 µg/L highest TUNEL-positive), all dosage groups: MDA boosted (membrane damage).	[74]
	PS/Spherical/2 µm/0.1 mg/L and 1.0 mg/L	Endpoints measured: Thyroid axis & maternal transfer: co-exposure increased maternal transfer of T3 and T4, and reduced thyroid hormones in the “F2 generation.”	63 d	Results: “At both concentrations” (0.1 and 1.0 mg/L), µ-PS exacerbated acetochlor-induced reductions in thyroid hormones and promoted maternal transfer.	[75]
	PE/Not disclosed/60 mg/L	Genotoxicity/cytotoxicity (blood cell biomarkers): Increased nuclear abnormalities, changes in erythrocyte and nuclear size/shape (mutagenic + cytotoxic signals), morphometric RBC nuclear/shape endpoints are shown in later figures (e.g., elongation/circularity).	10 d	Results: “At both concentrations” (0.1 and 1.0 mg/L), µ-PS exacerbated acetochlor-induced reductions in thyroid hormones and promoted maternal transfer. Genotoxicity (erythrocytes; comet assay): The DNA damage index was ~64% higher than control, and tail intensity increased by >60% relative to unexposed fish. Hydrogen peroxide (H2O2): PE-MP exposure was associated with higher H2O2 in brain (vs control) and higher H2O2 in liver (PE-MPs alone). In gills, PE-MP exposure showed reduced H2O2 versus control (this reduction was reported for PE-MPs alone).	[79]
	PSE/Spherical/100 µm/40.1 µg/L	Endocrine profile: Serum LH, FSH, and β-estradiol (E2) quantified by ELISA; kit ranges provided; absorbance read at 450 nm for E2; metabolomic/oxidative stress markers: Measured via glucometer; molecular biomarkers (qPCR).	21 d	Ovary testosterone: PS-MP increased ovarian testosterone by 75% vs. control; brain testosterone: PS-MP increased brain testosterone by 39.3% vs. control after 21 days. ovarian histology and oocyte maturation arrest (PCOS-like morphology): PS-MP ovaries: More developing immature (stage I/II) follicles and significantly fewer mature follicles (interpreted as chronic anovulation-like). Oxidative stress: MDA was significantly higher in PS-MP than control (direction/significance stated), with PS-MP MDA reported as 0.015 μM/mg ovarian tissue (LET: 0.104 μM/mg).	[76]
	PGA/~1 μm in diameter/1 mg/L & 100 mg/L	Gut barrier/intestinal permeability: Regulation downstream of Wnt/β-catenin; supported by qPCR, ELISA, tissue section analysis; gut microbiome: 16S rRNA sequencing; liver injury + histopathology and molecular assays (qPCR/ELISA) connecting gut disruption to systemic effects.	28 d	1 mg/L PGA MPs (28 d): Evidence of gut barrier disruption, microbiota dysbiosis, and behavioral/neurochemical disturbance (anxiety-like and impaired cognition/visual preference; altered 5-HT system). Wnt/β-catenin pathway genes (intestine): Low PGA reduced wnt-4a, high PGA increased wnt-4a and wnt-10b expression, while both doses reduced gsk3β and dkk1 expression, and high PGA increased β-catenin expression. Taxonomic changes (phylum-level trends): Reduced Proteobacteria, and increased Fusobacteria and Bacteroidetes after PGA exposure.	[47]
	PE/Spherical/25 μm 100 μg/L	Endocrine biomarkers (adults; sex hormones + VTG): Analytes: Estradiol (E2), testosterone (T), 11-keto testosterone (11-KT), vitellogenin (VTG); Genes: 17 HPGL-axis related genes listed in the paper (e.g., gnrh2/3, gnrhr2/3, lhβ, fshβ, fshr, lhr, star, cyp11a, cyp19a/b, erα/β, ar, vtg1, etc.); qPCR approach: SYBR Green real-time PCR; three biological replicates per treatment (as stated for this assay)	35 d	Pathological changes described including the loss of contact between oocyte membrane and follicular cell layer, yolk cell breakdown, and cell lysis; gnrhr2/gnrhr3: significantly reduced in all groups except MP group; additional gene-level guidance is mentioned in the text (e.g., cyp11a, lhr, erα, vtg downregulation; lhβ and erβ upregulation with MA significant for erβ; and MA-specific trends for cyp19a/cyp19b/star).	[78]
	PE/Microsphere/10–300 μm/0.1, 2, and 300 mg/L	Primary endpoints: Uptake, accumulation, and elimination of MPs (counts of fluorescent microspheres) in daphnia and zebrafish; fish tissues assessed for MPs: gill and digestive gland; feces collected for removal quantification in pathway experiment.	72 h	MP10 (0.1 mg/L): only a few particles at 1 h and only individual particles detectable at 12 h; interpreted by authors as inadvertent ingestion. (2 mg/L): accumulation peaked at 6 h with 163.18 particles/fish; then declined with time. MPs in gut (67.39% at 6 h; 53.85% at 12 h), while Exp2–Exp4 showed most MPs in feces; after 12 h in Exp2–Exp4, fecal proportions ranged ~87.86% to 98.22%.	[79]
	PE/Not disclosed/PE-MPs: VPE or APE at 1 mg/L (non-lethal; LC50 of MPs alone > 100 mg/L)	Oxidative stress: Enzymes: SOD, CAT; histopathology; immune gene expression: Genes: TLR-2, TLR-4, MyD88, NF-κB, NF-κB1, c-Rel, TNF-α, TNF-β, IL-1β; immune protein: NF-κB; gut microbiome.	35 d	Virgin polyethylene microplastics (VPE) and aged polyethylene microplastics (APE) caused significant changes in the intestinal microbial community. Both VPE and APE increased the relative abundance of Proteobacteria and reduced Fusobacteria. Oxidative stress responses: Lipid peroxidation (MDA) was increased in the APE group (~3.99%). Antioxidant enzyme activities indicated compensatory responses: Superoxide dismutase (SOD) activity was increased in VPE-treated fish. Catalase (CAT) activity indicated adaptive responses related to oxidative stress. Immune-related gene expression. Microplastic exposure increased intestinal immune signaling pathway markers: TLR-2, c-Rel APE treatment caused more immune activation than VPE. Intestinal barrier and inflammation. Indications of intestinal stress and inflammation related to microbiota imbalance and oxidative status.	[81]
	Virgin & Artificially weathered PP & PS/PS: ~15–36 µm PP: ~50–148 µm; After weathering: (≤230 µm)/Environmentally relevant (particle based): 2000, 20,000, 200,000 MP·L−1; High concentrations (mass based): 12.5, 25, 50, 100 mg·L−1	Malformations (edema, scoliosis, hemorrhage), heart rate, body length, swimming bladder, microplastic–chorion interaction.	96 h (outcomes recorded at 24/48/72/96 h; heart rate at 72 h; length at 96 h)	2000 MP·L−1: ↓ heart rate, ↓ body length. 20,000 MP·L−1: Sublethal growth effects. 200,000 MP·L−1: No linear increase in toxicity. 12.5–100 mg·L−1: No significant embryotoxicity.	[38]
	Weathered PE/Fragmented/32 µm/1 µg/L	Innate immune metrics: lysozyme, antimicrobial, antiprotease activity; hematology: differential counts + RBC indices (MCV/MCH/MCHC discussion indicates anemia typing); stress physiology: Plasma cortisol elevated (stress response).	40 d	Results 1 μg/L: Significant modulation of lysozyme, antimicrobial, antiprotease activity, plus altered blood differential counts; male fish more susceptible than females after chronic exposure; hematological interpretation suggests macrocytic-type anemia signatures; (MCV/MCH changes; MCHC decreased in both sexes after 40 d, with weaker change in females); cortisol increased, consistent with chronic stress from MP accumulation	[82]
Embryos	PE/Spherical/10–150 µm/100 mg/L, 500 mg/L, and 1000 mg/L	Cellular & physiological: Microplastic accumulation (fluorescence microscopy), excretion patterns, reactive oxygen species (ROS)—H2DCFDA staining, cell death—acridine orange staining. Molecular (qRT-PCR): Antioxidant genes: sod2, cat, hmox1, nfe2l2a, keap1a, DNA damage/apoptosis genes: puma, mdm2, tp53.	Up to 120 hpf (5 days); excretion observed up to 11 d	100 mg/L: Accumulation: Detected at ≥96 hpf (eye, gut, liver); ROS: ↑ ROS (moderate); cell death: ↑ apoptosis; gene expression: Early antioxidant gene upregulation. 500 mg/L: Accumulation: Increased accumulation; ROS: ↑↑ ROS; cell death: ↑↑ apoptosis; gene expression: downregulation of antioxidant genes. 1000 mg/L: Accumulation: Highest accumulation; ROS: ↑↑↑ ROS; cell death: ↑↑↑ apoptosis; gene expression: Strong oxidative stress & DNA damage response.	[83]
	Virgin & Chlorinated PS/Both Pristine and Chlorinated MPs ~5 µm/0.25 mg/L, 1.0 mg/L, and 4.0 mg/L	Intestinal histopathology, gut microbiota, oxidative stress biomarkers in gut (SOD, CAT, MDA), immune signaling (gene transcription), and nf-κb protein.	96–120 hpf	Gut microbiota: Chao1 index: increased significantly in VPE-only and APE-only groups vs. control; VPE-only higher than APE-only. Shannon index: Increased significantly in VPE-only and APE-only groups vs. control; VPE-only showed the largest increase (~2.88× control). After 7 days: SOD and CAT activities in “other treatment groups” (i.e., non-PTH-alone) increased significantly (range given 17.32–143.84%). After 14 days: CAT activity remained elevated in each treatment group vs. control (increase ~55.00–113.18%); MDA in the APE-only group was significantly increased by 3.99% vs. control. After 21 days: SOD activity in the PE-MPs treatment group returned to baseline levels; MDA content decreased in all treatment groups by ~17.03–57.70% vs. control. VPE-only (gene expression): TLR-2, MyD88, c-Rel, TNF-β, and IL-1β increased by 1.13–1.79× vs. control. APE-only (gene expression): TLR-2 and c-Rel upregulated (~2.10× and ~1.95×), while NF-κB1 and IL-1β downregulated (~0.61× and ~0.73×) vs. control. nf-κb protein content in gut: VPE-only and APE-only did not show a significant change vs. control (the significant change described is for penthiopyrad alone and for combined groups).	[84]
	PE/Microsphere/8.0 µm/50 µg/L and 500 µg/L	qRT-PCR gene expression: IGF-related: igf1, igf2a, igf2b, igfra, igfrb; GH-related: ghrh, gh1, ghra, ghrb.	72 h	Low MPs (50 µg/L) and low PFOS (0.02 µg/L) activated gene expression rapidly (short time window). High MPs (500 µg/L) and high PFOS (0.1 µg/L) activate genes rapidly and sustain elevated expression longer.	[85]
	PS/Spheres/0.1 μm diameter/0, 0.1, 1, 10, 50, 100 mg/L	Oxidative stress: ROS (DCFH-DA), antioxidant/related markers (later sections); apoptosis: Acridine orange staining; transcriptomics + metabolomics; DEG/GO/KEGG (cell cycle, retinol, ferroptosis, p53).	96 h	Oxidative stress: ROS production was measured in larvae following embryo-larval exposure, with a PS-MP-alone group added (“Larvae exposed to 1 mg/L PS”). Apoptosis: Acridine orange (AO) staining-based apoptosis analysis included a PS-MPs-alone group (“Larvae exposed to 1 mg/L PS”).	[49]
Embryos → Larvae	PS/Spherical/1 µm in diameter/0.1, 1, and 10 mg/L	Oxidative biomarkers: SOD, CAT, GPx, MDA, ROS	21 d	0.1 mg/L: Slight oxidative stress; mild increase in SOD and CAT activities; no behavioral alteration. 10 mg/L: High oxidative stress (↑MDA levels 2× control); reduced swimming velocity; histopathological changes in liver	[86]
	PE/Mean diameter 58.9 ± 4.52 µm/0.0, 12.5, 50 and 100 mg·L−1	Juveniles: Gastrointestinal retention and depuration of PE microplastics. Adults: Histology: Organs analyzed: Intestine, gills, liver. Adult: Genotoxicity and cytotoxicity: Micronucleus test, nuclear abnormalities, Comet assay (alkaline). Adults: Biochemical biomarkers: Acetylcholinesterase (AChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH).	Embryo: 96 h, Juvenile: 72 h, Adult: 96 h	Juveniles’ depuration: after exposure, the gastrointestinal tract eliminated the microplastics gradually; after 15 days in clean water (post-exposure), the intestinal lumen agglomerate disappeared (recovery test). Adult tissue distribution/histology: PE microplastics agglomerated with fecal content in the intestinal lumen and were detectable there, but were not observed in the intestinal wall/villi, nor in gill or liver. Genotoxicity/cytotoxicity: Micronucleus test showed no chromosome breaks/malsegregation and no nuclear abnormalities across exposure levels (p > 0.05). Comet assay: no increase in DNA break indices across exposure levels (p > 0.05), except the positive control (H2O2 0.1%, p < 0.05). Neurotoxicity-related biomarker (AChE): AChE activity in adult head showed significant differences at 50 and 100 mg/L versus control (*** p < 0.001) indicating highly significant, while tail AChE showed no difference (p > 0.05). LDH (tail): LDH activity was not modified versus control (p > 0.05). GST: body GST activity significantly decreased at 50 and 100 mg/L (* p < 0.05) which indicates statistically significant, while gill GST activity increased with a concentration–effect relationship (*** p < 0.001) with high significant.	[87]
	Fluorescent plastic microspheres/Spherical/1–5 μm/2 mg/L (~1.09 × 108 particles/L).	Gene expression: Neurogenesis/proliferation: sox2, pcna, ngn1, neuroD, olig2; motor neuron development: islet1, islet2a, islet2b; epigenetic regulation; dnmt1, dnmt3–dnmt8; related to antioxidant activity (sod1, cat), apoptosis (casp3, casp8, casp9), neurogenesis (pcna, sox2), and neurotransmitter systems (cholinergic, serotonergic, dopaminergic) Cellular and histopathology endpoints: Immunohistochemistry (PCNA, ISL1&2), stereological analysis of retina and brain, histopathology (retina, brain).	2 hpf to 14 dpf.	Virgin MPs (proprietary polymer): Inhibited GPx activity; upregulated sod1, casp8, casp9, casp3, th, and slc6a3 genes; increased AChE activity. Induced behavioral changes in mean speed and distance moved. Apoptosis: MPs alone significantly increased the expression of apoptosis-related genes (casp3, casp8, casp9).	[50]
	PS/Spherical/20 µm/2 mg/L	Thyroid axis parameters: T3, T4, TSH. Metabolomics: metabolites such as BHA, arachidonic acid and glycerophospholipid pathways.	7 d	Uptake/distribution: After 7 days, the 20 μm fluorescent polystyrene MPs were mainly found to be accumulated in the GI tract of the larvae (none were found in the controls). Thyroid axis endpoint: MPs did not significantly change T3. No significant difference in the results in metabolomic analysis.	[88]
	PS/Spherical/5 µm and 10 µm/1, 10, and 100 mg/L	Molecular: Oxidative stress biomarkers, antioxidant enzyme activities, lipid peroxidation, expression of heart-related genes. Histopathological: Heart tissue structure, cardiomyocyte alterations, apoptotic markers.	96 hpf	Oxidative damage and genotoxicity in heart tissue after dietary PS-MP exposure (21 days): Lipid peroxidation was higher in PS-MPs treated fish (reported as +528.5% vs. control), and DNA damage was higher (reported as ~100× higher vs. control). Autophagy markers: LC3 II/I ratio increased (reported as 2.2-fold higher) and SQSTM1/p62 decreased (reported as 2.8-fold lower) in PS-MPs treated fish vs. control. Apoptosis markers: Bax/Bcl-2 ratio increased (reported as 5.1-fold higher) and caspase-3 and caspase-9 increased (reported as 2.5-fold higher) in PS-MP-treated fish vs. control. Heart metabolomics (PS-MPs exposed vs. control): The metabolic profile of heart tissue was altered, with most metabolites reduced; pyruvic acid (+38%) and acetylcarnitine (+14%) increased, while TCA intermediates (e.g., succinic acid −75%, α-ketoglutaric acid −56%) and multiple amino acids were reduced.	[67]
	PS/Microbeads/5 ± 3 μm & 50 ± 3 μm/10 mg/L at 5 μm (MPs-5) or 50 μm (MPs-50)	Oxidative stress and oxidative damage genes: ROS, MDA, DNA damage markers, CAT; apoptosis-related gene expression such as p53, Bax, and Bcl-2; Dioxin-like marker genes (CYP1A1 and CYP1B1).	Up to 96 hpf	Oxidative stress and oxidative damage: increased ROS with associated increases in lipid peroxidation (MDA) and oxidative DNA damage marker (8-OHdG), and activation/induction of antioxidant enzymes like SOD and CAT. Apoptosis-related genes: p53 and Bax were upregulated and Bcl-2 was downregulated vs. controls and caspase 3, 8, 9 were upregulated in PS-MP-exposed treatments. At 5 µm PS-MPs: CYP1A1 and CYP1B1 significantly upregulated vs. control (CK). At 50 µm PS-MPs: No significant difference vs. control (CK) for these CYP responses.	[56]
	PP/Mixed fragments/11.9–44.6 µm/0 mg/L, 1 mg/L, 10 mg/L, 100 mg/L	Gut MP load; oxidative stress; ROS; SOD, CAT; neurotoxicity; acetylcholinesterase (AChE); Histopathology; Liver; Brain.	28 d	0 (control): No effect 1 mg/L: Mild ROS elevation, early antioxidant imbalance. 10 mg/L: Significant oxidative stress, liver histological damage, increased AChE activity. 100 mg/L: Severe oxidative stress, marked hepatic and neural injury, apoptosis of blood cells, high MP bioaccumulation in gut.	[89]
	PE/0–10 mm/ 0 mg/L, 10 mg/L, 100 mg/L, 1000 mg/L	Gut microbiota: qPCR at phylum level (all 4 groups) + 16S sequencing. Biochemical indicators (physiology): TG, GLU, TCHO, TBA, LDL, HDL, pyruvic acid, NEFA. Gene expression (glycolipid & phospholipid metabolism): RT-qPCR in 0/10/100/1000 mg/L groups. Metabolomics: Nontargeted LC-MS metabolomics only for Control vs. 1000 mg/L (6 parallels; 400 larvae/sample).	7 d	0 mg/L (control): Nontargeted LC-MS metabolomics only for control vs. 1000 mg/L (6 parallels; 400 larvae/sample). 10 mg/L: Minimal to lower significant. 100 mg/L: Microbiome (qPCR, phylum level): Firmicutes and Bacteroidetes significantly lower vs. control; Actinobacteria, β-Proteobacteria, γ-Proteobacteria significantly reduced vs. control (also true at 1000). 1000 mg/L: Microbiome (16S sequencing vs. control): alpha diversity shifts; OTUs decrease; clear separation in beta diversity (PCA), Proteobacteria/Chloroflexi/Fusobacteria ↑; Firmicutes/Bacteroidetes/Actinobacteria and others ↓, Genus-level dysbiosis (Aeromonas/Shewanella etc ↑; many beneficial taxa ↓), biochemical indicators: TG, TCHO, NEFA, TBA, GLU significantly increased; pyruvic acid significantly decreased; LDL/HDL show a decreasing trend, Glycolysis/glucose genes: PK, HK1, GK decreased significantly at 1000 mg/L; PEPCKc decreased at 100 and 1000.	[90]
	PS/Spherical/1 µm diameter/100 µg/L and 1000 µg/L	Gene expression (il1b, cat, sod).	4 hpf → 96–120 hpf.	100 µg/L: No effect on the gene expression. 1000 µg/L: Significant ↑ inflammatory gene (il1b), ↑ oxidative stress marker (cat).	[91]
	Not disclosed/~1 µm diameter/MPs suspended at 0.006%, 0.0045%, 0.003%, 0.0015% solids	Microcirculation/RBC velocity using high-speed CCD and micro-PIV analysis. Heart morphology.	Exposed for 3 days (1 dpf → 4 dpf)	The caudal artery (CA), systole–diastole cycle duration was elongated in MP3-ZF without changing RBC velocity. The duration and magnitude of RBC velocity at the dorsal artery (DA) were not significantly altered.	[51]
	PS/Not disclosed/1, 10, 100 µg/L	Endpoints measured: Cardiac development: heart rate (HR) at multiple hpf; cardiac morphology; SV–BA distance; histopathology. Thrombosis: thrombus in caudal vein in 100 µg/L MC-LR + PS-MPs/NPs group. Angiogenesis: Inhibited angiogenesis (DLAV/ISV loss) in Tg(kdrl:EGFP) model; amelioration by ASTA, oxidative stress + inflammation: ROS staining; MDA/SOD/GSH; IL-6/IL-8 mRNA by qRT-PCR at 168 hpf. Gene expression pathways: Cardiovascular development genes & calcium signaling pathway genes altered (heatmaps).	7 d	Results: At 96 & 168 hpf: MC-LR + PS-MPs → lower heart rate vs. control and MC-LR alone groups. 100 µg/L MC-LR + PS-MPs: thrombus observed; vascular loss; oxidative stress/inflammation increased; ASTA provides partial rescue.	[92]
	PGA, PLA, PBS, PHA, PBAT/No data/1 mg/L and 100 mg/L for each polymer	Thyroid axis/molecular endpoints: Hormones (ELISA): T3, T4, TSH in 120 hpf larvae (tested using 100 mg/L groups); Gene expression (qRT-PCR): HPT axis genes + eye/retina development genes (samples taken at 72 hpf; again, focusing on 100 mg/L groups for gene work).	3, 6, 10, 24, and 96 hpf	1 mg/L: Survival: Significantly decreased for PHA (1 mg/L) and PBAT (1 mg/L) at 96 hpf; body length/head area: Reduced in most low-dose groups except PGA 1 mg/L (which showed no significant reduction). Eye area: decreased in most low-dose groups except PGA 1 mg/L and PBAT 1 mg/L (no significant decrease reported for those two). Retina thickness (5 dpf): IPL decreased at PGA 1 mg/L (reported significant reduction); ONL decreased at PBS 1 mg/L and PBAT 1 mg/L; RGL decreased in all 1 mg/L groups (and also all 100 mg/L groups). Visually-mediated behavior (light–dark): In light, locomotor parameters were reduced in all treatment groups (interpreted as inhibited motor ability during light period. 100 mg/L: Early embryonic development: PBS, PHA, PBAT (100 mg/L) caused developmental delay at 3 hpf (PBS notably with embryonic cell mound defects). Retina & eye development gene expression: Eye/retina genes (e.g., pax6a, pax6b, rx1, gnat2, grk1b, opn1mw1) were abnormally altered; notably, PGA/PLA/PBAT 100 mg/L significantly decreased multiple eye/retina development genes. RGL thickness decreased in all groups, supporting retinal injury across polymers/doses. Thigmotaxis (3 dpf): Thigmotaxis increased significantly in PGA, PLA, PBS, PBAT at 100 mg/L, described as anxiety-like behavior. Visually-mediated behavior (5 dpf): PBS 100 mg/L is explicitly flagged as disrupting visually-mediated behavior (impaired light-to-dark response pattern). Thyroid hormones (ELISA, 100 mg/L only): TSH: No significant change overall. T3 and T4: Significantly decreased in PHA 100 mg/L and PBAT 100 mg/L; not significantly changed for PGA/PLA/PBS 100 mg/L. HPT axis gene disruption (100 mg/L groups): crh downregulated (notably PGA/PBS/PHA), tshβ upregulated in all groups. dio1 and ttr generally down; dio2 up with PLA 100 mg/L. TH synthesis genes: tg down in PBAT; nis down in PLA and PBAT; tpo up in PBAT. TH receptors: thrab increased in PGA/PBS/PHA/PBAT; thrb decreased in PLA but increased in PBS/PHA/PBAT.	[53]
	PS/Microsphere/5 μm/1 mg/L	Oxidative stress biomarkers: CAT, SOD, GPX activities; MDA level; ROS measurement (DCFH-DA assay described); TG level (lipid-related index) at 7 dpf.	2 h post-fertilization (hpf) and continued to 7 days post-fertilization (dpf).	Oxidative stress biomarker: CAT down by 1.55× (NPs) and 1.22× (MPs), SOD down by 1.76× (NPs) and 1.62× (MPs), GPX down by 1.96× (NPs) and 1.58× (MPs), ROS up by 1.20× (NPs) and 1.41× (MPs).	[57]
	PET & PE/Irregular fragments/Average 30–100 µm/1 mg/L & 10 mg/L	Enzymatic biomarkers (EROD, AChE), reproductive output (fecundity/fertility), and offspring (F1) fitness.	0 to 96- or 120-h post-fertilization (hpf)	Biomarkers: EROD and AChE activities were altered only in marine medaka, not zebrafish.	[55]
Larvae	PS/0.2, 1.0, and 10 µm/20 µg/mL for all particle sizes (0.2, 1.0, 10 µm)	Mortality; vascular development; CYP1A activity (EROD Assay). Oxidative stress (ROS); cell death (acridine orange staining).	Larvae: Chorion removed at 24 hpf, Exposure from 24–96 hpf (72 h post-initiation).	CYP1A activity (EROD, heart region): PS-MPs alone did not induce CYP1A activity. ROS in heart region: PSMPs alone were not significantly different from control for ROS. Cell death (acridine orange signal, heart region): PSMPs alone did not affect cell death.	[93]
	PS & PVC/Spherical/PS (Spherical ~7.0 µm mean size) PVC (Spherical ~3.8 µm mean size)/Both MPS 20 mg L−1	Histopathology: Intestinal MP deposition, inflammation, pleural effusion, cellular damage (H&E staining). Molecular and biochemical: Oxidative stress genes (sod, cat, gpx1a, gpx4a, gstt1a), inflammation genes (tnf-α, il-6), apoptosis/p53 pathway genes (tp53, casp3, rrm2), development & metabolism genes (egr2, egr4, fosab, fosb, cyp3a65).	10 d	MPs visibly accumulated in intestinal tract, Inflammatory infiltration and pleural effusion observed, PS caused slightly more damage than PVC. Gene expression: MPs alone significantly upregulated oxidative stress genes, PS induced stronger oxidative stress than PVC. PS MPs were more toxic than PVC MPs to zebrafish larvae, Joint toxicity depended strongly on MP polymer type; Oxidative stress was the central mechanism linking mortality, behavior, histopathology, and gene expression.	[58]
	PP/Irregular fragments/8–10 µm/0 mg/L (control), 1 mg/L, 10 mg/L, 100 mg/L	Cytotoxicity and tissue damage: Liver histopathology, intestinal histopathology, cellular vacuolization, inflammatory infiltration. Oxidative stress biomarkers: Reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA). Apoptosis and molecular markers: Caspase-3 activity, bax, bcl-2 gene transcription, p53 transcription. Neurotoxicity and metabolic endpoints: Acetylcholinesterase (AChE) & ATP content.	96 h (acute exposure)	Control: Normal liver and intestinal morphology, baseline ROS, enzyme activity, and gene transcription. 1 mg/L: Slight increase in ROS, no significant histological damage, Minor antioxidant response. 10 mg/L: Significant ROS elevation, decreased SOD and CAT, increased MDA, mild liver vacuolization, increased bax and caspase-3 expression, reduced ATP levels. 100 mg/L: Severe oxidative stress, strong lipid peroxidation, marked liver and intestinal tissue damage; high apoptosis activation, suppressed AChE activity, significant metabolic disruption.	[94]
	PS/5 µm/1 mg/L	Toxicity endpoints (larval): Oxidative stress gene expression such as GSH/GSSG.	7 d	Oxidative-stress–linked metabolite: Glutathione (GSH) increased in the MP-only group (fold change 1.17). GSH/GSSG: No significant difference in the results.	[95]
	PS/~25 μm in diameter/250 μg/L	Apoptosis markers: bax/bcl2, caspase3; AO staining brain.	Early dpf stages	Apoptosis-related genes (whole larvae): PS-MPs upregulated the bax/bcl2 ratio and caspase3 expression at 1 dpf, but not at 3 dpf and 5 dpf. Apoptosis staining (brain region): At 5 dpf, there were no apoptotic bodies in the larval brain regions exposed to PS-MPs.	[62]
	Virgin & UV-aged PA/Irregular fragments/~5 µm (mean particle diameter)/0, 10, 100, and 1000 µg/L	Intestinal health: Intestinal morphology (H&E staining), Goblet cell number, Tight junction–related gene expression. Lipid absorption: Oil Red O staining and Triglyceride content. Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.), and Intestinal barrier genes (zo-1, claudin).	2 d post-fertilization (dpf) → 10 dpf	Mild changes in the intestinal structure were noticed, such as slight desquamation of enterocytes and vacuolation in the intestinal mucosa. Oxidative stress responses were triggered, as evidenced by enhanced production of reactive oxygen species and changes in the activity of antioxidant enzymes. There was impairment in lipid metabolism, as evidenced by decreased expression of lipid metabolites (triglycerides, diglycerides, monoglycerides, cholesterol esters, phospholipids). Inhibition of lipoprotein lipase activity: Decreased expression of genes involved in fat digestion and absorption. Decreased triglyceride and cholesterol concentrations, suggesting impaired triglyceride and cholesterol absorption.	[48]
	Virgin & Photo-aged PS/Virgin: 10 μm Photo-aged: 6.5 μm/0.1–100 μg/L for both V-PS and P-PS.	Oxidative stress biomarkers: SOD, CAT, GST, MDA; Molecular: expression of neurotransmission genes (ache, drd3, 5ht2c, gat1, etc.) and oxidative stress genes (cat1, sod1, gpx1a, gstrl, etc.).	1 hfp to 120 hpf	Oxidative stress: Antioxidant enzymes + MDA significantly altered at 10–100 μg/L P-PS. As well as the SOD, CAT, and GST are significantly altered.	[47]
	PS/Virgin & Photo-aged/Spherical beads/1 µm/0, 1, 10, and 100 µg/L both Virgin & Photo-aged	Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.), and Intestinal barrier genes (zo-1, claudin).	96 h post-fertilization (hpf)	Photo-aged PS MPs: caused intestinal epithelial damage, Reduced goblet cell number, disrupted intestinal barrier genes, severely impaired lipid absorption.	[59]

Abbreviations: PS—polystyrene; PP—polypropylene; PE—polyethylene; PES—polyester; PVC—polyvinyl chloride; PGA—polyglycolic acid; PBAT—polybutylene adipate terephthalate; PLA—polylactic acid; PHA—polyhydroxyalkanoate; PBS—polybutylene succinate; PET—polyethylene terephthalate; PA—polyamide; MPs—microplastics; hpf—hours post-fertilization; dpf—days post-fertilization; h—hours; d—days; w—weeks; DNMT—DNA methyltransferase; PCNA—proliferating cell nuclear antigen; 5-HT—5-hydroxytryptamine; GABA—gamma-aminobutyric acid; DA—dopamine; Ach—acetylcholine; AChE—acetylcholinesterase; ChAT—choline acetyltransferase; ChE—cholinesterase; AgNP—silver nanoparticles; GFP—green fluorescent protein; SOD—Superoxide dismutase; CAT—Catalase; GPx—Glutathione peroxidase; MDA—Malondialdehyde; ROS—Reactive oxygen species; GR—Glutathione reductase; GI—Gastrointestinal; PCoA—Principal Coordinate Analysis; LDH—Lactate dehydrogenase; GST—Glutathione S-transferase; CYP1A—Cytochrome P450 1A; EROD—Ethoxyresorufin-O-deethylase; HMOX1—Heme oxygenase 1; nfe212a—Nuclear factor erythroid 2-related factor 2a; keap1a—Kelch-like ECH-associated protein 1a; tp53—tumor protein p53; NEFA—Non-esterified fatty acids; LDL—Low-density lipoprotein; T-CHO—Total cholesterol; TG—Triglycerides; GSH—Reduced glutathione; Mn-sod—Manganese superoxide dismutase; Bcl2—B-cell lymphoma 2; UV—ultra-violet; PPM—parts per million; ↑—increased; ↑↑—moderately increased; ↑↑↑—markedly/substantially increased; ↓—decreased; →—progression.

Table 2. Physiological effects of pristine/virgin/unspecifies and UV/photo-aging in MP exposure.

Life Stage	Plastic Characterization (Type/Shape/Size/Concentrations)	Endpoints	Exposure Time	Effects	Ref.
Adult	PE/Spherical/Average diameter of 40 ± 10 µm/100 µg/L	Oxidative stress biomarkers: SOD, CAT, GPx, MDA, T-AOC.	21 d	100 µg/L: Slight oxidative stress (↑ SOD, CAT activities); minor intestinal villi damage.	[80]
	PS/Spherical beads/1 μm & 3 μm/0.01, 0.1, 1.0, 10.0 mg/L	Cardiac physiology (heart rate at 72 h); Redox homeostasis/oxidative stress biomarkers.	96 h (4 days post-fertilization)	↑ in heart rate both concentrations with more strongly at 10 mg/L. ↓ ROS content at 10 mg/L; ↑ Lipid hydroperoxides but more significant in 10 mg/L. Antioxidant enzymes: GPX: Unaffected; GR: ↑ significantly only in 1 mg/L; SOD: ↑ significantly only in 10 mg/L.	[37]
	Virgin & UV PP/Virgin: 33.2 ± 14.4 μm UV: 20.8 ± 10.5 μm/50 mg/L both V & UV	Histopathology: Intestinal villi length, mucus secretion, goblet cell number, vacuolization, ciliary defects. Gene expression: Sod1, IL-1β, CLDN5, Oclna, ZO-1. Enzyme biomarkers: SOD, IL-1β (inflammation), and D-lactate (intestinal permeability). Gut microbiome: Alpha diversity (Chao1, Shannon), Beta diversity (PCoA, UniFrac), OTU abundance, taxonomic shifts at phylum & genus levels, KEGG pathway analysis using PICRUSt & MinPath.	14 d	Tissue distribution and accumulation: Both V & UV weathered PP particles were detected in the GI tract, liver, and gills. Peak GI burden (day 3): PP = 383.4 ± 50.3 particles; UV-PP = 2053.7 ± 371.4 particles. Excretion kinetics (GI): Elimination half-life PP = 0.78 d; UV-PP = 0.38 d; after 5 days of depuration, both MPs reached 99.9% excretion. Intestinal histopathology: Exposure to PP and UV-PP produced mucosal damage characterized by structural damage, vacuolization, ciliary defects, mucus secretion, and reduced goblet cells. Goblet cells: Decreased by 34% (PP) and 51% (UV-PP). Oxidative stress/inflammation markers (gut): SOD mRNA upregulated significantly in UV-PP; il-1β mRNA upregulated significantly in both PP and UV-PP vs. S.C; SOD and IL-1β enzyme-level changes were consistent with gene expression patterns. Tight junction/barrier markers: cldn5 and zo-1 transcription downregulated in PP and UV-PP; D-lactate (D-Lac) increased. Gut microbiota diversity and composition: Shannon index increased significantly in the PP group; PCoA indicated microbiome shifts after PP and UV-PP exposure. Differential taxa (reported comparisons): Rhizobium, Gemmobacter, and Cloacibacterium were significantly higher in PP than UV-PP; Luteolibacter and Rodobacter were significantly higher in UV-PP; Porphyromonadaceae and Aeromonas tended to decrease in MP-exposed groups.	[41]
	Virgin MPs (propriety polymer)/Spherical/1–5 μm/2 mg/L (~1.09 × 108 particles/L)	Biochemical biomarkers: ROS, LPO, SOD, CAT, GPx, GST, GR, GSH/GSSG, LDH, AChE, and MT. Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.), and intestinal barrier genes (zo-1, claudin).	2 hpf to 14 dpf	Significant inhibition of acetylcholinesterase (AChE) activity compared with control, indicating neurotoxicity. AChE inhibition showed correlation with mortality increase and reduced growth. Other biomarkers such as ROS, LPO, SOD, CAT, GST, GR, GSH/GSSG, LDH, significantly affect the overall physiological capacity of the zebrafish.	[65]
	PE & PES/Fragmented beads/Average size of 180 ± 210 µm/1 mg/L both PE & PES	Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.) and intestinal barrier genes (zo-1, claudin). Oxidative stress: ROS production, antioxidant enzymes (SOD, CAT), lipid peroxidation (MDA).	96 h (4 days) acute exposure	PE exposure: Significantly altered lipid metabolism in the intestine and liver, and perturbation of lipid metabolism, fatty acid metabolism, vitamin metabolism, TCA cycle, and amino acid metabolism. PES exposure: Upregulated metabolites included phosphocholine and 2-lysophosphatidylcholine; and downregulation metabolites included triglyceride, 13-HDoHE, n-triacontanol, and phosphatidylserine.	[43]
	Propriety polymer (composition undisclosed/Spherical/1–5 µm/2 mg/L	Molecular responses: Oxidative stress-related genes, detoxification-related genes. Metabolomics: Whole-body metabolomics, LC-MS/MS (QTOF) untargeted metabolomics.	30 d	MPs (2 mg/L): ↑ Metallothionein (MT), ↑ tph1a (serotonin synthesis gene), ↓ LDH (metabolic alteration). Gut microbiota: Increased; Fusobacteria, and Protobacteria; Decreased: Firmicute; Clear dysbiosis in both PE and PES groups.	[64]
	PS/Microbeads/200 µm, 40 µm, 10 µm/100 µg/L	Biochemical biomarkers: ALT, AST (hepatic injury), SOD, CAT, GSH (oxidative stress), integrated biomarker response (IBR).	30 d	MP exposure resulted in liver pathological changes, wherein liver damage included the presence of ballooning of the hepatocytes (vacuolization), nuclear abnormalities (including the formation of pyknotic or peripherally located nuclei), and the presence. CAT activities decreased with the decrease of MPs size, and the smallest MP group showed a significant decrease in CAT under single exposure. In the MP single-exposure groups, SOD activity increased with decreasing plastic size. GSH content significantly rose under the single MP treatments, but the magnitude of GSH increase showed a decline with a decrease in MPs size.	[73]
	PS/5 µm/2 mg/L	Oxidative stress biomarkers: Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), malondialdehyde (MDA). Histopathology: Liver tissue, hematoxylin & eosin (H&E) staining, hepatocyte vacuolization, cellular degeneration, histopathological scoring. Gene expression (qRT-PCR): Metallothionein (mt2), antioxidant-related genes, apoptosis-related genes.	14 d	Gills (Histopathology): There were no histopathological changes in the gills of the fish that were exposed to not contaminated. Intestine (Histopathology): Intestinal changes are minor, including cracking of villi, caused by MPs alone. Liver (histopathology): There was no obvious impact of MPs alone on histopathology.	[44]
	PE & PES/Fragments/PE mean size: 180 ± 210 µm PES mean length: 350 ± 220 µm/0.2 mg/L, 1 mg/L both PE & PES	Whole-body untargeted LC-MS/MS metabolomics to quantify MP-associated shifts in molecular metabolites and enriched metabolic pathways. Gut microbiome physiology and gut microbiota profiling after MP exposure.	30 d	PE-specific metabolomic changes: 0.2 mg/L and 1 mg/L overlapped with minimal separation, while both separated from controls. Both concentrations had no significant different and does not change metabolomics of zebrafish. Gut microbiome (16S metagenomics): Community composition: Dominant phyla included Proteobacteria, Fusobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Verrucomicrobia; Fusobacteria increased (0.3–11.7%) in microplastics-exposed groups vs. control, while Proteobacteria decreased (0.4–9.0%) across exposures vs. control. Potential pathogens were reported only in exposed groups (examples: Mycobacterium in PES2; Aeromonas in PE1/PE2). Diversity: Alpha diversity indices (observed OTU, Shannon, Faith FD, Simpson) were reported as not significantly altered across exposure groups. PE: Physical epithelial damage, oxidative stress, inflammation, disrupted membrane and energy metabolism. PES: Fibers interfered more strongly with gut microbiota interactions, lipid digestion, and endocrine-related metabolism.	[45]
	PVC & PP/Irregular fragments/5–50 µm/PVC MPs (100 µg/L), and PP MPs (100 µg/L)	Intestinal histopathology (H&E); intestinal inflammation biomarkers/gene expressions: TNF-α levels and IL-1β gene expression. Oxidative stress biomarkers/antioxidant-related expression; intestinal metabolomics; and intestinal microbiota	21 d	Intestinal histopathology (H&E): Control intestines showed intact villi shape and regularly arranged epithelial cells. PVC-only and PP-only groups showed slight shedding of intestinal villi. Intestinal inflammation biomarkers/gene expression: TNF-α levels significantly increased in all treatment groups except the cadmium-only group; this includes PVC-only and PP-only. IL-1β gene expression significantly increased in all treatment groups; this includes PVC-only and PP-only. Oxidative stress biomarkers/antioxidant-related expression: No remarkable shifts in SOD activity or MDA levels were reported after exposure to PVC alone or PP alone. PP (but not PVC) significantly increased GPx expression (detoxification-related). Intestinal metabolomics: PVC-only: 32 significant differential metabolites downregulated and 24 upregulated vs. control. PP-only: 85 significant differential metabolites downregulated and 10 upregulated vs. control. KEGG pathway enrichment: both PVC-only and PP-only showed disturbances in purine metabolism and arginine/proline metabolism. PVC-only additionally disrupted primary bile acid biosynthesis and arachidonic acid metabolism. PP-only additionally affected arginine biosynthesis and glutamate metabolism. Intestinal microbiota: At the phylum level, Firmicutes relative abundance increased in PVC-only and PP-only groups. Proteobacteria relative abundance increased in all treatment groups, including PVC-only and PP-only.	[65]
	PS/Spherical beads/2 μm diameter/0.44 mg/L (~108 items/L)	Oxidative biomarkers: SOD, CAT, GSH, and MDA	7 d	Ocular oxidative stress biomarkers (eyes; ELISA; normalized to protein): MP exposure showed no significant main effect on SOD, CAT, GSH, or MDA in the model analysis (p > 0.1). GSH specifically: The MP-only group had significantly higher ocular GSH than the control group. MDA: No significant difference was detected in ocular MDA among groups.	[66]
	PS/Spherical beads/2–4 µm diameter/440 µg/L	Oxidative stress biomarkers: SOD, CAT activities, MDA content, and protein normalization. Histopathology: Villus number, villus height and width, and intestinal wall thickness; and gut microbiota analysis.	21 d	PS beads were observed in the gut at day 1 and day 21 (control showed only autofluorescence). PS distributed along anterior–middle–posterior gut and tended to accumulate in the mid-posterior gut and/or be excreted with fecal pellets. Oxidative stress/anti-oxidant response: CAT activity was significantly increased in the PS group compared with the control group (and AMI group). SOD activity was not reported as significantly increased in the PS group (significance was reported for the PS + AMI group vs. control/PS/AMI). MDA was not reported as significantly increased in the PS group (the significant change reported was a decrease in the AMI group vs. control/PS). Morphometrics: Villus width was significantly increased in the PS-MP group; intestinal wall thickness was significantly decreased in the PS-MP group. The number of villi showed decreasing tendency in the PS-MP group. Gut microbiota: PS-MPs showed an increasing tendency in Proteobacteria, which includes Exiguobacterium, Candidatus paracaedibacter, and Staphylococcus.	[69]
	PS/Spherical beads/5 µm diameter/20 µg/L	Hepatic biochemical parameters: Energy metabolism: glucose, pyruvate, Lipid metabolism: TG, T-CHO, LDL-C, NEFA, oxidative stress: SOD, CAT, GSH, MDA Glycolipid metabolism genes: Glycolysis/gluconeogenesis: Gk, Hk1, Pepckc, β-oxidation: Aco, Cpt1, Ppar-α, Lipid synthesis: Acc, Fas, Ppar-γ, Lipid transport: Apo, Fabp6. Oxidative stress genes: Mn-sod, Cu/Zn-sod, Cat, Nrf2, Keap1, Gpx, Bcl2; Inflammatory genes: IL-1β, IL-6, IL-8, TNF-α, IFN, C3, IL-10.	21 d	Hepatic metabolism: PS: ↓ pyruvate, TG, T-CHO. PS specific significantly changes: Hepatic pyruvate, TG, and T-CHO with significant decreased; PS had no significant effect on NEFA. Hepatic glycolipid metabolism genes: Based from the results, PS had no significant effect on glycolipid metabolism genes. Oxidative biomarkers: CAT activity had a significant increase in PS exposure vs. control.; the GSH content was reduced after 21 days of exposure; oxidative stress-related mRNA such as Mn-sod, Nrf2, Keap1, Gpx, and Bcl2 mRNA levels were significantly inhibited in PS-MPs.	[46]
	Virgin & Photo-aged PLA/~100 µm/5 mg·L−1	Female reproductive endpoints: Gonadosomatic index (GSI), ovary histology (H&E), sex hormones: Testosterone (T), estradiol (E2), Emphasis on E2/T; ovary metabolomics.	5 w	DPLA > UPLA toxicity, ovarian structural damage, disrupted steroid hormones, altered metabolomic pathways, offspring: ↑ mortality, ↓ hatching, ↓ body length.	[48]
	PE/Spherical/146.2 ± 8.9 µm/5 µg/L and 50 µg/L	Oxidative stress & antioxidant biomarkers: Catalase (CAT), glutathione-S-transferase (GST), lipid peroxidation (MDA); ion regulation: Na+/K+-ATPase activity (gills).	10 d & 20 d	5 µg/L: 10 days: ↓ CAT and GST (liver); 20 days: ↑ GST activity (adaptive response), ↑ lipid peroxidation (brain). 50 µg/L: Stronger oxidative stress than 5 µg/L, significant ↑ MDA (brain), marked ↑ Na+/K+-ATPase activity in gills.	[70]
	PS/Spherical beads/1 µm/30 mg/L	MP distribution (gills vs. gut), gut histopathology, oxidative stress enzyme, liver metabolomics, gut microbiota (16S rRNA), sex hormones, and reproductive output.	Chronic (multi-week).	Females accumulated more PS, gut microbiota dysbiosis stronger in females, altered hepatic lipid & energy metabolism, reduced egg production, clear sex-specific toxicity.	[71]
	PS/Fragmented/5 µm/20 & 200 mg/L	Cd accumulation (liver, gut, gill), oxidative stress enzymes, histopathology, inflammatory gene expression.	Chronic (multi-week).	MPs enhanced Cd bioaccumulation, there was an increased trend in oxidative damage and inflammation synergistically increased, MPs acted as toxic vector.	[72]
	PS/Fragmented/5 µm/LMPs: 0.1, 1, 10, 50, 100 mg/L; SMPs: 0.1, 1, 10, 50, 100 mg/L	Antioxidant enzymes: SOD, CAT, GPx, lipid peroxidation (MDA).	96 h post-exposure	SOD activity: At day 4, PS-MP exposure significantly increased hepatic SOD. CAT activity: At day 4, CAT activity was significantly higher and remained significantly higher at day 8. GPx: At day 4, GPx activity was significantly higher and remained significantly higher at day 8. Lipid peroxidation (MDA): PS-MPs had no significant effect on hepatic MDA at day 4 but evidently reduced at day 8.	[49]
	PS/Not disclosed/0, 50, 500 µg/L	Gene expression: qPCR normalization to β-actin, ovarian histology: (oocyte stage scoring; H&E), oxidative stress markers: MDA (lipid peroxidation), SOD, CAT.	60 d	50 and 500 µg/L: Decreased ovarian SOD/CAT activity (oxidative stress), increased NO, increased apoptosis; dose response noted (500 µg/L highest TUNEL-positive), all dosage groups: MDA boosted (membrane damage).	[74]
	PS/Spherical/2 µm/0.1 mg/L and 1.0 mg/L	Endpoints measured: Thyroid axis & maternal transfer: co-exposure increased maternal transfer of T3 and T4, and reduced thyroid hormones in the “F2 generation.”	63 d	Results: “At both concentrations” (0.1 and 1.0 mg/L), µ-PS exacerbated acetochlor-induced reductions in thyroid hormones and promoted maternal transfer.	[75]
	PE/Not disclosed/60 mg/L	Genotoxicity/cytotoxicity (blood cell biomarkers): Increased nuclear abnormalities, changes in erythrocyte and nuclear size/shape (mutagenic + cytotoxic signals), morphometric RBC nuclear/shape endpoints are shown in later figures (e.g., elongation/circularity).	10 d	Results: “At both concentrations” (0.1 and 1.0 mg/L), µ-PS exacerbated acetochlor-induced reductions in thyroid hormones and promoted maternal transfer. Genotoxicity (erythrocytes; comet assay): The DNA damage index was ~64% higher than control, and tail intensity increased by >60% relative to unexposed fish. Hydrogen peroxide (H2O2): PE-MP exposure was associated with higher H2O2 in brain (vs control) and higher H2O2 in liver (PE-MPs alone). In gills, PE-MP exposure showed reduced H2O2 versus control (this reduction was reported for PE-MPs alone).	[79]
	PSE/Spherical/100 µm/40.1 µg/L	Endocrine profile: Serum LH, FSH, and β-estradiol (E2) quantified by ELISA; kit ranges provided; absorbance read at 450 nm for E2; metabolomic/oxidative stress markers: Measured via glucometer; molecular biomarkers (qPCR).	21 d	Ovary testosterone: PS-MP increased ovarian testosterone by 75% vs. control; brain testosterone: PS-MP increased brain testosterone by 39.3% vs. control after 21 days. ovarian histology and oocyte maturation arrest (PCOS-like morphology): PS-MP ovaries: More developing immature (stage I/II) follicles and significantly fewer mature follicles (interpreted as chronic anovulation-like). Oxidative stress: MDA was significantly higher in PS-MP than control (direction/significance stated), with PS-MP MDA reported as 0.015 μM/mg ovarian tissue (LET: 0.104 μM/mg).	[76]
	PGA/~1 μm in diameter/1 mg/L & 100 mg/L	Gut barrier/intestinal permeability: Regulation downstream of Wnt/β-catenin; supported by qPCR, ELISA, tissue section analysis; gut microbiome: 16S rRNA sequencing; liver injury + histopathology and molecular assays (qPCR/ELISA) connecting gut disruption to systemic effects.	28 d	1 mg/L PGA MPs (28 d): Evidence of gut barrier disruption, microbiota dysbiosis, and behavioral/neurochemical disturbance (anxiety-like and impaired cognition/visual preference; altered 5-HT system). Wnt/β-catenin pathway genes (intestine): Low PGA reduced wnt-4a, high PGA increased wnt-4a and wnt-10b expression, while both doses reduced gsk3β and dkk1 expression, and high PGA increased β-catenin expression. Taxonomic changes (phylum-level trends): Reduced Proteobacteria, and increased Fusobacteria and Bacteroidetes after PGA exposure.	[47]
	PE/Spherical/25 μm 100 μg/L	Endocrine biomarkers (adults; sex hormones + VTG): Analytes: Estradiol (E2), testosterone (T), 11-keto testosterone (11-KT), vitellogenin (VTG); Genes: 17 HPGL-axis related genes listed in the paper (e.g., gnrh2/3, gnrhr2/3, lhβ, fshβ, fshr, lhr, star, cyp11a, cyp19a/b, erα/β, ar, vtg1, etc.); qPCR approach: SYBR Green real-time PCR; three biological replicates per treatment (as stated for this assay)	35 d	Pathological changes described including the loss of contact between oocyte membrane and follicular cell layer, yolk cell breakdown, and cell lysis; gnrhr2/gnrhr3: significantly reduced in all groups except MP group; additional gene-level guidance is mentioned in the text (e.g., cyp11a, lhr, erα, vtg downregulation; lhβ and erβ upregulation with MA significant for erβ; and MA-specific trends for cyp19a/cyp19b/star).	[78]
	PE/Microsphere/10–300 μm/0.1, 2, and 300 mg/L	Primary endpoints: Uptake, accumulation, and elimination of MPs (counts of fluorescent microspheres) in daphnia and zebrafish; fish tissues assessed for MPs: gill and digestive gland; feces collected for removal quantification in pathway experiment.	72 h	MP10 (0.1 mg/L): only a few particles at 1 h and only individual particles detectable at 12 h; interpreted by authors as inadvertent ingestion. (2 mg/L): accumulation peaked at 6 h with 163.18 particles/fish; then declined with time. MPs in gut (67.39% at 6 h; 53.85% at 12 h), while Exp2–Exp4 showed most MPs in feces; after 12 h in Exp2–Exp4, fecal proportions ranged ~87.86% to 98.22%.	[79]
	PE/Not disclosed/PE-MPs: VPE or APE at 1 mg/L (non-lethal; LC50 of MPs alone > 100 mg/L)	Oxidative stress: Enzymes: SOD, CAT; histopathology; immune gene expression: Genes: TLR-2, TLR-4, MyD88, NF-κB, NF-κB1, c-Rel, TNF-α, TNF-β, IL-1β; immune protein: NF-κB; gut microbiome.	35 d	Virgin polyethylene microplastics (VPE) and aged polyethylene microplastics (APE) caused significant changes in the intestinal microbial community. Both VPE and APE increased the relative abundance of Proteobacteria and reduced Fusobacteria. Oxidative stress responses: Lipid peroxidation (MDA) was increased in the APE group (~3.99%). Antioxidant enzyme activities indicated compensatory responses: Superoxide dismutase (SOD) activity was increased in VPE-treated fish. Catalase (CAT) activity indicated adaptive responses related to oxidative stress. Immune-related gene expression. Microplastic exposure increased intestinal immune signaling pathway markers: TLR-2, c-Rel APE treatment caused more immune activation than VPE. Intestinal barrier and inflammation. Indications of intestinal stress and inflammation related to microbiota imbalance and oxidative status.	[81]
	Virgin & Artificially weathered PP & PS/PS: ~15–36 µm PP: ~50–148 µm; After weathering: (≤230 µm)/Environmentally relevant (particle based): 2000, 20,000, 200,000 MP·L−1; High concentrations (mass based): 12.5, 25, 50, 100 mg·L−1	Malformations (edema, scoliosis, hemorrhage), heart rate, body length, swimming bladder, microplastic–chorion interaction.	96 h (outcomes recorded at 24/48/72/96 h; heart rate at 72 h; length at 96 h)	2000 MP·L−1: ↓ heart rate, ↓ body length. 20,000 MP·L−1: Sublethal growth effects. 200,000 MP·L−1: No linear increase in toxicity. 12.5–100 mg·L−1: No significant embryotoxicity.	[38]
	Weathered PE/Fragmented/32 µm/1 µg/L	Innate immune metrics: lysozyme, antimicrobial, antiprotease activity; hematology: differential counts + RBC indices (MCV/MCH/MCHC discussion indicates anemia typing); stress physiology: Plasma cortisol elevated (stress response).	40 d	Results 1 μg/L: Significant modulation of lysozyme, antimicrobial, antiprotease activity, plus altered blood differential counts; male fish more susceptible than females after chronic exposure; hematological interpretation suggests macrocytic-type anemia signatures; (MCV/MCH changes; MCHC decreased in both sexes after 40 d, with weaker change in females); cortisol increased, consistent with chronic stress from MP accumulation	[82]
Embryos	PE/Spherical/10–150 µm/100 mg/L, 500 mg/L, and 1000 mg/L	Cellular & physiological: Microplastic accumulation (fluorescence microscopy), excretion patterns, reactive oxygen species (ROS)—H2DCFDA staining, cell death—acridine orange staining. Molecular (qRT-PCR): Antioxidant genes: sod2, cat, hmox1, nfe2l2a, keap1a, DNA damage/apoptosis genes: puma, mdm2, tp53.	Up to 120 hpf (5 days); excretion observed up to 11 d	100 mg/L: Accumulation: Detected at ≥96 hpf (eye, gut, liver); ROS: ↑ ROS (moderate); cell death: ↑ apoptosis; gene expression: Early antioxidant gene upregulation. 500 mg/L: Accumulation: Increased accumulation; ROS: ↑↑ ROS; cell death: ↑↑ apoptosis; gene expression: downregulation of antioxidant genes. 1000 mg/L: Accumulation: Highest accumulation; ROS: ↑↑↑ ROS; cell death: ↑↑↑ apoptosis; gene expression: Strong oxidative stress & DNA damage response.	[83]
	Virgin & Chlorinated PS/Both Pristine and Chlorinated MPs ~5 µm/0.25 mg/L, 1.0 mg/L, and 4.0 mg/L	Intestinal histopathology, gut microbiota, oxidative stress biomarkers in gut (SOD, CAT, MDA), immune signaling (gene transcription), and nf-κb protein.	96–120 hpf	Gut microbiota: Chao1 index: increased significantly in VPE-only and APE-only groups vs. control; VPE-only higher than APE-only. Shannon index: Increased significantly in VPE-only and APE-only groups vs. control; VPE-only showed the largest increase (~2.88× control). After 7 days: SOD and CAT activities in “other treatment groups” (i.e., non-PTH-alone) increased significantly (range given 17.32–143.84%). After 14 days: CAT activity remained elevated in each treatment group vs. control (increase ~55.00–113.18%); MDA in the APE-only group was significantly increased by 3.99% vs. control. After 21 days: SOD activity in the PE-MPs treatment group returned to baseline levels; MDA content decreased in all treatment groups by ~17.03–57.70% vs. control. VPE-only (gene expression): TLR-2, MyD88, c-Rel, TNF-β, and IL-1β increased by 1.13–1.79× vs. control. APE-only (gene expression): TLR-2 and c-Rel upregulated (~2.10× and ~1.95×), while NF-κB1 and IL-1β downregulated (~0.61× and ~0.73×) vs. control. nf-κb protein content in gut: VPE-only and APE-only did not show a significant change vs. control (the significant change described is for penthiopyrad alone and for combined groups).	[84]
	PE/Microsphere/8.0 µm/50 µg/L and 500 µg/L	qRT-PCR gene expression: IGF-related: igf1, igf2a, igf2b, igfra, igfrb; GH-related: ghrh, gh1, ghra, ghrb.	72 h	Low MPs (50 µg/L) and low PFOS (0.02 µg/L) activated gene expression rapidly (short time window). High MPs (500 µg/L) and high PFOS (0.1 µg/L) activate genes rapidly and sustain elevated expression longer.	[85]
	PS/Spheres/0.1 μm diameter/0, 0.1, 1, 10, 50, 100 mg/L	Oxidative stress: ROS (DCFH-DA), antioxidant/related markers (later sections); apoptosis: Acridine orange staining; transcriptomics + metabolomics; DEG/GO/KEGG (cell cycle, retinol, ferroptosis, p53).	96 h	Oxidative stress: ROS production was measured in larvae following embryo-larval exposure, with a PS-MP-alone group added (“Larvae exposed to 1 mg/L PS”). Apoptosis: Acridine orange (AO) staining-based apoptosis analysis included a PS-MPs-alone group (“Larvae exposed to 1 mg/L PS”).	[49]
Embryos → Larvae	PS/Spherical/1 µm in diameter/0.1, 1, and 10 mg/L	Oxidative biomarkers: SOD, CAT, GPx, MDA, ROS	21 d	0.1 mg/L: Slight oxidative stress; mild increase in SOD and CAT activities; no behavioral alteration. 10 mg/L: High oxidative stress (↑MDA levels 2× control); reduced swimming velocity; histopathological changes in liver	[86]
	PE/Mean diameter 58.9 ± 4.52 µm/0.0, 12.5, 50 and 100 mg·L−1	Juveniles: Gastrointestinal retention and depuration of PE microplastics. Adults: Histology: Organs analyzed: Intestine, gills, liver. Adult: Genotoxicity and cytotoxicity: Micronucleus test, nuclear abnormalities, Comet assay (alkaline). Adults: Biochemical biomarkers: Acetylcholinesterase (AChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH).	Embryo: 96 h, Juvenile: 72 h, Adult: 96 h	Juveniles’ depuration: after exposure, the gastrointestinal tract eliminated the microplastics gradually; after 15 days in clean water (post-exposure), the intestinal lumen agglomerate disappeared (recovery test). Adult tissue distribution/histology: PE microplastics agglomerated with fecal content in the intestinal lumen and were detectable there, but were not observed in the intestinal wall/villi, nor in gill or liver. Genotoxicity/cytotoxicity: Micronucleus test showed no chromosome breaks/malsegregation and no nuclear abnormalities across exposure levels (p > 0.05). Comet assay: no increase in DNA break indices across exposure levels (p > 0.05), except the positive control (H2O2 0.1%, p < 0.05). Neurotoxicity-related biomarker (AChE): AChE activity in adult head showed significant differences at 50 and 100 mg/L versus control (*** p < 0.001) indicating highly significant, while tail AChE showed no difference (p > 0.05). LDH (tail): LDH activity was not modified versus control (p > 0.05). GST: body GST activity significantly decreased at 50 and 100 mg/L (* p < 0.05) which indicates statistically significant, while gill GST activity increased with a concentration–effect relationship (*** p < 0.001) with high significant.	[87]
	Fluorescent plastic microspheres/Spherical/1–5 μm/2 mg/L (~1.09 × 108 particles/L).	Gene expression: Neurogenesis/proliferation: sox2, pcna, ngn1, neuroD, olig2; motor neuron development: islet1, islet2a, islet2b; epigenetic regulation; dnmt1, dnmt3–dnmt8; related to antioxidant activity (sod1, cat), apoptosis (casp3, casp8, casp9), neurogenesis (pcna, sox2), and neurotransmitter systems (cholinergic, serotonergic, dopaminergic) Cellular and histopathology endpoints: Immunohistochemistry (PCNA, ISL1&2), stereological analysis of retina and brain, histopathology (retina, brain).	2 hpf to 14 dpf.	Virgin MPs (proprietary polymer): Inhibited GPx activity; upregulated sod1, casp8, casp9, casp3, th, and slc6a3 genes; increased AChE activity. Induced behavioral changes in mean speed and distance moved. Apoptosis: MPs alone significantly increased the expression of apoptosis-related genes (casp3, casp8, casp9).	[50]
	PS/Spherical/20 µm/2 mg/L	Thyroid axis parameters: T3, T4, TSH. Metabolomics: metabolites such as BHA, arachidonic acid and glycerophospholipid pathways.	7 d	Uptake/distribution: After 7 days, the 20 μm fluorescent polystyrene MPs were mainly found to be accumulated in the GI tract of the larvae (none were found in the controls). Thyroid axis endpoint: MPs did not significantly change T3. No significant difference in the results in metabolomic analysis.	[88]
	PS/Spherical/5 µm and 10 µm/1, 10, and 100 mg/L	Molecular: Oxidative stress biomarkers, antioxidant enzyme activities, lipid peroxidation, expression of heart-related genes. Histopathological: Heart tissue structure, cardiomyocyte alterations, apoptotic markers.	96 hpf	Oxidative damage and genotoxicity in heart tissue after dietary PS-MP exposure (21 days): Lipid peroxidation was higher in PS-MPs treated fish (reported as +528.5% vs. control), and DNA damage was higher (reported as ~100× higher vs. control). Autophagy markers: LC3 II/I ratio increased (reported as 2.2-fold higher) and SQSTM1/p62 decreased (reported as 2.8-fold lower) in PS-MPs treated fish vs. control. Apoptosis markers: Bax/Bcl-2 ratio increased (reported as 5.1-fold higher) and caspase-3 and caspase-9 increased (reported as 2.5-fold higher) in PS-MP-treated fish vs. control. Heart metabolomics (PS-MPs exposed vs. control): The metabolic profile of heart tissue was altered, with most metabolites reduced; pyruvic acid (+38%) and acetylcarnitine (+14%) increased, while TCA intermediates (e.g., succinic acid −75%, α-ketoglutaric acid −56%) and multiple amino acids were reduced.	[67]
	PS/Microbeads/5 ± 3 μm & 50 ± 3 μm/10 mg/L at 5 μm (MPs-5) or 50 μm (MPs-50)	Oxidative stress and oxidative damage genes: ROS, MDA, DNA damage markers, CAT; apoptosis-related gene expression such as p53, Bax, and Bcl-2; Dioxin-like marker genes (CYP1A1 and CYP1B1).	Up to 96 hpf	Oxidative stress and oxidative damage: increased ROS with associated increases in lipid peroxidation (MDA) and oxidative DNA damage marker (8-OHdG), and activation/induction of antioxidant enzymes like SOD and CAT. Apoptosis-related genes: p53 and Bax were upregulated and Bcl-2 was downregulated vs. controls and caspase 3, 8, 9 were upregulated in PS-MP-exposed treatments. At 5 µm PS-MPs: CYP1A1 and CYP1B1 significantly upregulated vs. control (CK). At 50 µm PS-MPs: No significant difference vs. control (CK) for these CYP responses.	[56]
	PP/Mixed fragments/11.9–44.6 µm/0 mg/L, 1 mg/L, 10 mg/L, 100 mg/L	Gut MP load; oxidative stress; ROS; SOD, CAT; neurotoxicity; acetylcholinesterase (AChE); Histopathology; Liver; Brain.	28 d	0 (control): No effect 1 mg/L: Mild ROS elevation, early antioxidant imbalance. 10 mg/L: Significant oxidative stress, liver histological damage, increased AChE activity. 100 mg/L: Severe oxidative stress, marked hepatic and neural injury, apoptosis of blood cells, high MP bioaccumulation in gut.	[89]
	PE/0–10 mm/ 0 mg/L, 10 mg/L, 100 mg/L, 1000 mg/L	Gut microbiota: qPCR at phylum level (all 4 groups) + 16S sequencing. Biochemical indicators (physiology): TG, GLU, TCHO, TBA, LDL, HDL, pyruvic acid, NEFA. Gene expression (glycolipid & phospholipid metabolism): RT-qPCR in 0/10/100/1000 mg/L groups. Metabolomics: Nontargeted LC-MS metabolomics only for Control vs. 1000 mg/L (6 parallels; 400 larvae/sample).	7 d	0 mg/L (control): Nontargeted LC-MS metabolomics only for control vs. 1000 mg/L (6 parallels; 400 larvae/sample). 10 mg/L: Minimal to lower significant. 100 mg/L: Microbiome (qPCR, phylum level): Firmicutes and Bacteroidetes significantly lower vs. control; Actinobacteria, β-Proteobacteria, γ-Proteobacteria significantly reduced vs. control (also true at 1000). 1000 mg/L: Microbiome (16S sequencing vs. control): alpha diversity shifts; OTUs decrease; clear separation in beta diversity (PCA), Proteobacteria/Chloroflexi/Fusobacteria ↑; Firmicutes/Bacteroidetes/Actinobacteria and others ↓, Genus-level dysbiosis (Aeromonas/Shewanella etc ↑; many beneficial taxa ↓), biochemical indicators: TG, TCHO, NEFA, TBA, GLU significantly increased; pyruvic acid significantly decreased; LDL/HDL show a decreasing trend, Glycolysis/glucose genes: PK, HK1, GK decreased significantly at 1000 mg/L; PEPCKc decreased at 100 and 1000.	[90]
	PS/Spherical/1 µm diameter/100 µg/L and 1000 µg/L	Gene expression (il1b, cat, sod).	4 hpf → 96–120 hpf.	100 µg/L: No effect on the gene expression. 1000 µg/L: Significant ↑ inflammatory gene (il1b), ↑ oxidative stress marker (cat).	[91]
	Not disclosed/~1 µm diameter/MPs suspended at 0.006%, 0.0045%, 0.003%, 0.0015% solids	Microcirculation/RBC velocity using high-speed CCD and micro-PIV analysis. Heart morphology.	Exposed for 3 days (1 dpf → 4 dpf)	The caudal artery (CA), systole–diastole cycle duration was elongated in MP3-ZF without changing RBC velocity. The duration and magnitude of RBC velocity at the dorsal artery (DA) were not significantly altered.	[51]
	PS/Not disclosed/1, 10, 100 µg/L	Endpoints measured: Cardiac development: heart rate (HR) at multiple hpf; cardiac morphology; SV–BA distance; histopathology. Thrombosis: thrombus in caudal vein in 100 µg/L MC-LR + PS-MPs/NPs group. Angiogenesis: Inhibited angiogenesis (DLAV/ISV loss) in Tg(kdrl:EGFP) model; amelioration by ASTA, oxidative stress + inflammation: ROS staining; MDA/SOD/GSH; IL-6/IL-8 mRNA by qRT-PCR at 168 hpf. Gene expression pathways: Cardiovascular development genes & calcium signaling pathway genes altered (heatmaps).	7 d	Results: At 96 & 168 hpf: MC-LR + PS-MPs → lower heart rate vs. control and MC-LR alone groups. 100 µg/L MC-LR + PS-MPs: thrombus observed; vascular loss; oxidative stress/inflammation increased; ASTA provides partial rescue.	[92]
	PGA, PLA, PBS, PHA, PBAT/No data/1 mg/L and 100 mg/L for each polymer	Thyroid axis/molecular endpoints: Hormones (ELISA): T3, T4, TSH in 120 hpf larvae (tested using 100 mg/L groups); Gene expression (qRT-PCR): HPT axis genes + eye/retina development genes (samples taken at 72 hpf; again, focusing on 100 mg/L groups for gene work).	3, 6, 10, 24, and 96 hpf	1 mg/L: Survival: Significantly decreased for PHA (1 mg/L) and PBAT (1 mg/L) at 96 hpf; body length/head area: Reduced in most low-dose groups except PGA 1 mg/L (which showed no significant reduction). Eye area: decreased in most low-dose groups except PGA 1 mg/L and PBAT 1 mg/L (no significant decrease reported for those two). Retina thickness (5 dpf): IPL decreased at PGA 1 mg/L (reported significant reduction); ONL decreased at PBS 1 mg/L and PBAT 1 mg/L; RGL decreased in all 1 mg/L groups (and also all 100 mg/L groups). Visually-mediated behavior (light–dark): In light, locomotor parameters were reduced in all treatment groups (interpreted as inhibited motor ability during light period. 100 mg/L: Early embryonic development: PBS, PHA, PBAT (100 mg/L) caused developmental delay at 3 hpf (PBS notably with embryonic cell mound defects). Retina & eye development gene expression: Eye/retina genes (e.g., pax6a, pax6b, rx1, gnat2, grk1b, opn1mw1) were abnormally altered; notably, PGA/PLA/PBAT 100 mg/L significantly decreased multiple eye/retina development genes. RGL thickness decreased in all groups, supporting retinal injury across polymers/doses. Thigmotaxis (3 dpf): Thigmotaxis increased significantly in PGA, PLA, PBS, PBAT at 100 mg/L, described as anxiety-like behavior. Visually-mediated behavior (5 dpf): PBS 100 mg/L is explicitly flagged as disrupting visually-mediated behavior (impaired light-to-dark response pattern). Thyroid hormones (ELISA, 100 mg/L only): TSH: No significant change overall. T3 and T4: Significantly decreased in PHA 100 mg/L and PBAT 100 mg/L; not significantly changed for PGA/PLA/PBS 100 mg/L. HPT axis gene disruption (100 mg/L groups): crh downregulated (notably PGA/PBS/PHA), tshβ upregulated in all groups. dio1 and ttr generally down; dio2 up with PLA 100 mg/L. TH synthesis genes: tg down in PBAT; nis down in PLA and PBAT; tpo up in PBAT. TH receptors: thrab increased in PGA/PBS/PHA/PBAT; thrb decreased in PLA but increased in PBS/PHA/PBAT.	[53]
	PS/Microsphere/5 μm/1 mg/L	Oxidative stress biomarkers: CAT, SOD, GPX activities; MDA level; ROS measurement (DCFH-DA assay described); TG level (lipid-related index) at 7 dpf.	2 h post-fertilization (hpf) and continued to 7 days post-fertilization (dpf).	Oxidative stress biomarker: CAT down by 1.55× (NPs) and 1.22× (MPs), SOD down by 1.76× (NPs) and 1.62× (MPs), GPX down by 1.96× (NPs) and 1.58× (MPs), ROS up by 1.20× (NPs) and 1.41× (MPs).	[57]
	PET & PE/Irregular fragments/Average 30–100 µm/1 mg/L & 10 mg/L	Enzymatic biomarkers (EROD, AChE), reproductive output (fecundity/fertility), and offspring (F1) fitness.	0 to 96- or 120-h post-fertilization (hpf)	Biomarkers: EROD and AChE activities were altered only in marine medaka, not zebrafish.	[55]
Larvae	PS/0.2, 1.0, and 10 µm/20 µg/mL for all particle sizes (0.2, 1.0, 10 µm)	Mortality; vascular development; CYP1A activity (EROD Assay). Oxidative stress (ROS); cell death (acridine orange staining).	Larvae: Chorion removed at 24 hpf, Exposure from 24–96 hpf (72 h post-initiation).	CYP1A activity (EROD, heart region): PS-MPs alone did not induce CYP1A activity. ROS in heart region: PSMPs alone were not significantly different from control for ROS. Cell death (acridine orange signal, heart region): PSMPs alone did not affect cell death.	[93]
	PS & PVC/Spherical/PS (Spherical ~7.0 µm mean size) PVC (Spherical ~3.8 µm mean size)/Both MPS 20 mg L−1	Histopathology: Intestinal MP deposition, inflammation, pleural effusion, cellular damage (H&E staining). Molecular and biochemical: Oxidative stress genes (sod, cat, gpx1a, gpx4a, gstt1a), inflammation genes (tnf-α, il-6), apoptosis/p53 pathway genes (tp53, casp3, rrm2), development & metabolism genes (egr2, egr4, fosab, fosb, cyp3a65).	10 d	MPs visibly accumulated in intestinal tract, Inflammatory infiltration and pleural effusion observed, PS caused slightly more damage than PVC. Gene expression: MPs alone significantly upregulated oxidative stress genes, PS induced stronger oxidative stress than PVC. PS MPs were more toxic than PVC MPs to zebrafish larvae, Joint toxicity depended strongly on MP polymer type; Oxidative stress was the central mechanism linking mortality, behavior, histopathology, and gene expression.	[58]
	PP/Irregular fragments/8–10 µm/0 mg/L (control), 1 mg/L, 10 mg/L, 100 mg/L	Cytotoxicity and tissue damage: Liver histopathology, intestinal histopathology, cellular vacuolization, inflammatory infiltration. Oxidative stress biomarkers: Reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA). Apoptosis and molecular markers: Caspase-3 activity, bax, bcl-2 gene transcription, p53 transcription. Neurotoxicity and metabolic endpoints: Acetylcholinesterase (AChE) & ATP content.	96 h (acute exposure)	Control: Normal liver and intestinal morphology, baseline ROS, enzyme activity, and gene transcription. 1 mg/L: Slight increase in ROS, no significant histological damage, Minor antioxidant response. 10 mg/L: Significant ROS elevation, decreased SOD and CAT, increased MDA, mild liver vacuolization, increased bax and caspase-3 expression, reduced ATP levels. 100 mg/L: Severe oxidative stress, strong lipid peroxidation, marked liver and intestinal tissue damage; high apoptosis activation, suppressed AChE activity, significant metabolic disruption.	[94]
	PS/5 µm/1 mg/L	Toxicity endpoints (larval): Oxidative stress gene expression such as GSH/GSSG.	7 d	Oxidative-stress–linked metabolite: Glutathione (GSH) increased in the MP-only group (fold change 1.17). GSH/GSSG: No significant difference in the results.	[95]
	PS/~25 μm in diameter/250 μg/L	Apoptosis markers: bax/bcl2, caspase3; AO staining brain.	Early dpf stages	Apoptosis-related genes (whole larvae): PS-MPs upregulated the bax/bcl2 ratio and caspase3 expression at 1 dpf, but not at 3 dpf and 5 dpf. Apoptosis staining (brain region): At 5 dpf, there were no apoptotic bodies in the larval brain regions exposed to PS-MPs.	[62]
	Virgin & UV-aged PA/Irregular fragments/~5 µm (mean particle diameter)/0, 10, 100, and 1000 µg/L	Intestinal health: Intestinal morphology (H&E staining), Goblet cell number, Tight junction–related gene expression. Lipid absorption: Oil Red O staining and Triglyceride content. Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.), and Intestinal barrier genes (zo-1, claudin).	2 d post-fertilization (dpf) → 10 dpf	Mild changes in the intestinal structure were noticed, such as slight desquamation of enterocytes and vacuolation in the intestinal mucosa. Oxidative stress responses were triggered, as evidenced by enhanced production of reactive oxygen species and changes in the activity of antioxidant enzymes. There was impairment in lipid metabolism, as evidenced by decreased expression of lipid metabolites (triglycerides, diglycerides, monoglycerides, cholesterol esters, phospholipids). Inhibition of lipoprotein lipase activity: Decreased expression of genes involved in fat digestion and absorption. Decreased triglyceride and cholesterol concentrations, suggesting impaired triglyceride and cholesterol absorption.	[48]
	Virgin & Photo-aged PS/Virgin: 10 μm Photo-aged: 6.5 μm/0.1–100 μg/L for both V-PS and P-PS.	Oxidative stress biomarkers: SOD, CAT, GST, MDA; Molecular: expression of neurotransmission genes (ache, drd3, 5ht2c, gat1, etc.) and oxidative stress genes (cat1, sod1, gpx1a, gstrl, etc.).	1 hfp to 120 hpf	Oxidative stress: Antioxidant enzymes + MDA significantly altered at 10–100 μg/L P-PS. As well as the SOD, CAT, and GST are significantly altered.	[47]
	PS/Virgin & Photo-aged/Spherical beads/1 µm/0, 1, 10, and 100 µg/L both Virgin & Photo-aged	Molecular responses: Lipid metabolism genes (fabp, apoa1, etc.), and Intestinal barrier genes (zo-1, claudin).	96 h post-fertilization (hpf)	Photo-aged PS MPs: caused intestinal epithelial damage, Reduced goblet cell number, disrupted intestinal barrier genes, severely impaired lipid absorption.	[59]

Abbreviations: PS—polystyrene; PP—polypropylene; PE—polyethylene; PES—polyester; PVC—polyvinyl chloride; PGA—polyglycolic acid; PBAT—polybutylene adipate terephthalate; PLA—polylactic acid; PHA—polyhydroxyalkanoate; PBS—polybutylene succinate; PET—polyethylene terephthalate; PA—polyamide; MPs—microplastics; hpf—hours post-fertilization; dpf—days post-fertilization; h—hours; d—days; w—weeks; DNMT—DNA methyltransferase; PCNA—proliferating cell nuclear antigen; 5-HT—5-hydroxytryptamine; GABA—gamma-aminobutyric acid; DA—dopamine; Ach—acetylcholine; AChE—acetylcholinesterase; ChAT—choline acetyltransferase; ChE—cholinesterase; AgNP—silver nanoparticles; GFP—green fluorescent protein; SOD—Superoxide dismutase; CAT—Catalase; GPx—Glutathione peroxidase; MDA—Malondialdehyde; ROS—Reactive oxygen species; GR—Glutathione reductase; GI—Gastrointestinal; PCoA—Principal Coordinate Analysis; LDH—Lactate dehydrogenase; GST—Glutathione S-transferase; CYP1A—Cytochrome P450 1A; EROD—Ethoxyresorufin-O-deethylase; HMOX1—Heme oxygenase 1; nfe212a—Nuclear factor erythroid 2-related factor 2a; keap1a—Kelch-like ECH-associated protein 1a; tp53—tumor protein p53; NEFA—Non-esterified fatty acids; LDL—Low-density lipoprotein; T-CHO—Total cholesterol; TG—Triglycerides; GSH—Reduced glutathione; Mn-sod—Manganese superoxide dismutase; Bcl2—B-cell lymphoma 2; UV—ultra-violet; PPM—parts per million; ↑—increased; ↑↑—moderately increased; ↑↑↑—markedly/substantially increased; ↓—decreased; →—progression.

5. Neurobehavior and Its Corresponding Effects on Zebrafish Exposed to Microplastics

Neurobehavioral results across studies show that microplastic exposure can significantly alter locomotor activity in larval and juvenile zebrafish, with responses strongly influenced by particle concentration, size, and exposure duration. In zebrafish larvae, exposure to PS and PVC MPs at 20 mg/L for 10 days resulted in clear locomotor impairment (Table 3). In this experiment, PS exposure produced higher mortality (~10–20%) and markedly suppressed swimming activity, whereas PVC exposure produced moderate locomotor inhibition and lower mortality, indicating polymer-specific differences in behavioral toxicity [57]. Also, zebrafish larvae exposed to PS MPs with a particle size of approximately 25 µm at concentrations of 25 and 250 µg/L showed no significant changes in locomotor behavior, including movement distance, average velocity, and maximum acceleration during light–dark cycle assays [80]. In comparison, zebrafish exposed to PS microbeads (5 ± 3 µm) at 10 mg/L resulted in 31.3% larval malformation and 11.1% mortality, with associated reductions in swimming performance due to developmental abnormalities affecting tail morphology and neuromuscular coordination [56].

Also, neurotransmitter regulation and cholinergic function were strongly affected by MP exposure under defined experimental conditions. In the study of [65], zebrafish larvae exposed to virgin PS MPs with a particle size of ~1 µm at concentrations of 1, 10, and 100 µg/L from 1 hpf to 120 hpf showed significant alterations in several neurotransmitters, including serotonin (5-HT), dopamine (DA), γ-aminobutyric acid (GABA), and acetylcholine (ACh). These exposures also altered cholinergic enzyme activities, including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and cholinesterase (ChE), indicating disruption of neurotransmission pathways associated with neural development and behavioral regulation (Figure 10).

Additional experiments demonstrate that the direction of AChE modulation varies depending on polymer type, particle size, and exposure conditions. For instance, zebrafish larvae exposed to spherical MPs (1–5 µm) at 2 mg/L from 2 hpf to 14 dpf showed significant inhibition of AChE activity, accompanied by increased reactive oxygen species (ROS), lipid peroxidation (LPO), and altered antioxidant biomarkers including SOD, CAT, GPx, GST, and GR, linking oxidative stress to neurophysiological impairment [65]. And neurochemical alterations were observed when zebrafish were exposed to PS particles of 50 nm and 45 µm from 4 to 120 hpf, where AChE activity decreased while antioxidant enzymes including CAT and GPx increased, together with elevated glutathione (GSH) levels and reduced body length (~6.1%), suggesting that oxidative stress-mediated neuronal disruption contributed to impaired neurodevelopment and locomotor activity (Table 3) [89].

Additionally, gene expression studies revealed that MP exposure can disrupt neurodevelopmental and neurofunctional processes in zebrafish under specific particle properties and exposure conditions. In the study of Li et al. [48], zebrafish embryos exposed to artificially aged PS MPs with a particle size of ~1 µm at concentrations of 0.1, 1, 10, and 100 µg/L exhibited significant alterations in neuronal development. After exposure until 120 hpf, aged PS particles reduced motor neuron fluorescence intensity from 238 ± 2.58 AU in controls to approximately 229 ± 2.53 AU at 100 µg/L, indicating impaired motor neuron formation. Gene expression analysis showed downregulation of neurodevelopment-related genes such as gabra1 and manf and upregulation of neural progenitor and structural genes including nestin, gfap, alpha-tubulin, and mbp, suggesting dysregulated neuronal differentiation and glial activation during early nervous system formation.

In addition, exposure of zebrafish embryos to fluorescent plastic microspheres (1–5 µm) at 2 mg/L from 2 hpf to 14 dpf resulted in abnormal neurodevelopmental signaling in retinal tissues. These particles accumulated in the retina and caused increased PCNA-positive proliferating cells, indicating compensatory cell proliferation. However, transcription of key neurogenesis genes such as sox2, neuroD, and olig2 was significantly downregulated, and DNA methyltransferase (DNMT) expression was altered, suggesting that microplastics can interfere with both neural differentiation pathways and epigenetic regulatory mechanisms controlling neurodevelopment [49]. Additional evidence of neuronal damage was observed in experiments where zebrafish exposed to PS MPs (~5 µm) at concentrations of 1–100 mg/L exhibited significant activation of apoptosis pathways. Neural tissues showed upregulation of apoptosis-related genes including casp3, casp8, and casp9 together with oxidative stress biomarkers, indicating activation of programmed cell death pathways in response to MP-induced cellular damage [86].

Cardiovascular and neurobehavioral integration was also documented under defined exposure conditions. In the study of La Pietra et al. [37], zebrafish embryos exposed to spherical PS microbeads with particle sizes of 1 and 5 µm at concentrations of 10, 100, and 1000 µg/L for 96 hpf showed measurable cardiotoxic effects. At 1000 µg/L, embryos exhibited a ~13% reduction in heart rate relative to controls, accompanied by developmental delay and increased deformities, including ~22% malformed embryos with pericardial edema and spinal curvature. These cardiovascular impairments were associated with reduced locomotor performance and altered physiological activity during larval development [36]. Cardiac oxidative injury was further demonstrated in chronic exposure experiments using spherical PS microplastics (~5 µm) at concentrations of 1, 10, and 100 mg/L. Metabolomic and biochemical analyses revealed severe oxidative damage in cardiac tissue, including a 529% increase in lipid peroxidation markers and an approximately 100-fold increase in DNA oxidative damage indicators compared with control groups. These responses were accompanied by significant activation of apoptosis-related pathways, including increased Bax/Bcl-2 ratio and elevated expression of caspase-3 and caspase-9, indicating oxidative injury and programmed cell death in cardiac tissues [88].

Co-exposure studies demonstrated that MPs could intensify toxicity of other environmental contaminants. Zebrafish embryos exposed to spherical PS MPs (~1 µm) at concentrations of 1, 10, and 100 µg/L together with microcystin-LR (MC-LR) showed pronounced cardiovascular disruption. Histological examination revealed thrombus formation in caudal blood vessels, inhibition of angiogenesis, and increased vascular abnormalities compared with single exposures (Table 3). These combined exposures also produced elevated oxidative stress biomarkers and inflammatory responses, including increased expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), indicating synergistic toxicity between microplastics and cyanobacterial toxins [63]. Chronic exposure in zebrafish microplastic studies generally refers to long-term exposure periods extending beyond early embryonic stages, typically ≥21 days to several weeks, during which organisms experience continuous or repeated exposure to microplastics at environmentally relevant or experimental concentrations. Chronic exposure designs are commonly used to evaluate endocrine disruption, metabolic alterations, microbiota shifts, and behavioral outcomes that cannot be detected in short-term acute toxicity assays conducted during early embryogenesis (e.g., 24–96 hpf exposure windows) [46,77,79].

Under these chronic conditions, endocrine–neurobehavioral interactions have been reported. In the study of Li et al. [92], exposed to PS MPs (~2 µm diameter) at concentrations of 0.1 and 1 mg/L for 63 days exhibited disruption of thyroid hormone regulation. The exposure altered circulating triiodothyronine (T3) and thyroxine (T4) levels and enhanced maternal transfer of thyroid hormones to embryos, which subsequently influenced developmental outcomes and behavioral responses in offspring. These endocrine alterations indicate that long-term microplastic exposure can interfere with the hypothalamic–pituitary–thyroid (HPT) axis, a key regulator of neural development and behavioral regulation [79]. Additional chronic experiments demonstrated interactions between gut physiology and behavioral regulation.

Zebrafish exposed to PS MPs (~5 µm) at 20 µg/L for 21 days developed intestinal barrier disruption, microbiota dysbiosis, and metabolic disturbances. Microbiome analyses showed significant shifts in dominant bacterial taxa, including increased Proteobacteria and altered Fusobacteria and Firmicutes abundance, which coincided with behavioral changes such as increased anxiety-like responses and reduced exploratory activity in locomotor assays. These responses were linked to intestinal inflammation and barrier impairment, indicating involvement of the gut–brain axis in behavioral dysfunction [77]. Bioaccumulation characteristics affect the intensity of neurobehavioral toxicity. Smaller and degraded particles produced stronger biological responses than larger or pristine particles. In the study of Kim et al. 2022 [81], zebrafish larvae exposed to photo-aged PS MPs (~1 µm diameter) at concentrations of 0.1, 1, 10, and 100 µg/L showed stronger developmental and neurodevelopmental impairment than larvae exposed to virgin PS at the same concentrations.

Aged PS exposure reduced motor neuron fluorescence intensity from 238 ± 2.58 AU in controls to 229 ± 2.53 AU at 100 µg/L, together with dysregulation of neurodevelopment-related genes including gabra1 and manf, indicating impaired neuronal differentiation and signaling [68]. Finally, MP exposure alters monoaminergic signaling pathways, including serotonin (5-HT), dopamine (DA), and γ-aminobutyric acid (GABA). These neurotransmitters regulate locomotion, anxiety behavior, and neural development. Changes in neurotransmitter levels occur when oxidative stress disrupts enzymes involved in synthesis and metabolism or when MPs interfere with neuronal gene expression controlling neurotransmitter transport and receptor signaling [61,95]. And MPs can interfere with endocrine signaling pathways such as the hypothalamic–pituitary–thyroid (HPT) and hypothalamic–pituitary–gonadal (HPG) axes.

Changes in hormones including thyroxine (T4), triiodothyronine (T3), testosterone, and estradiol affect neuronal differentiation, metabolism, and behavior. MPs may act as endocrine disruptors by adsorbing endocrine-active contaminants or by altering gene expression related to steroidogenesis and thyroid regulation [96,97]. Also, MP ingestion often leads to intestinal barrier damage and microbiota dysbiosis. Shifts in microbial communities (e.g., increased Proteobacteria and altered Firmicutes/Fusobacteria) can modify microbial metabolite production and immune signaling [62]. These gut-derived signals influence neural function through the gut–brain axis, contributing to anxiety-like behavior, altered locomotion, and cognitive dysfunction [98].

Table 3. Behavioral effects of pristine/virgin/unspecifies and UV/photo-aging in MP exposure.

Life Stage	Plastic Characterization (Type/Shape/Size/Concentrations)	Endpoints	Exposure Time	Effects	Ref.
Adult	PS/Irregular fragments/~400 µm/0.5 mg/L	Behavioral parameters: Latency to first MP capture, capture frequency, spitting frequency, swallowing ratio (%), feeding duration, time in feeding zone, swimming activity, speed, total distance moved.	10 m per trial	Latency to first capture: Most zebrafish started MP capture within ~10 s of adding MPs. Shy zebrafish showed longer latency than bold zebrafish (not significant). Capture frequency: Bold zebrafish captured MPs significantly more often than shy zebrafish on exposure days (p < 0.05). Spitting behavior: Zebrafish often spat MPs out after capture. About 30–47% of total capture events happened in the first minute. Spitting frequency was positively correlated with capture frequency. Swallowing ratio: About 40–60% of captured MPs were swallowed. Bold fish had higher effective exposure because of higher capture frequency. Swimming activity during feeding: Zebrafish showed immediate increase in activity after MP addition. Bold zebrafish showed significantly higher levels of feeding activity (activity %, total distance, average speed) than shy zebrafish on days 2–3 (p < 0.05). Bold zebrafish spent significantly more time in feeding areas (upper water layer). Ingestion/egestion behavior: Bold zebrafish ingested more MPs than shy zebrafish when exposed to MPs only (significant on day 2). Very few MPs were left in intestines after 3 days, suggesting efficient egestion.	[96]
	Propriety polymer (composition undisclosed/Spherical/1–5 µm/2 mg/L	Behavior: Locomotor activity (Open Field Test), anxiety (Light-Dark Test), and social.	30 d	Locomotor activity (open field test): Mean speed: Markedly reduced in MP group (584 ± 33.5 cm/min) compared to control (696 ± 27.1 cm/min) (p < 0.01). Total distance moved: Significantly reduced in MP group (2863.4 ± 1.8 cm) compared to control (3445.8 ± 234.4 cm) (p = 0.0395). Absolute turn angle: Not significantly different between MP group and control. Time inactive: Significantly increased in MP group (10.4 ± 0.3%) compared to control (3.0 ± 0.3%) (p < 0.0001). Time in center zone: no significant difference between MP group and control. Anxiety-like behavior (light–dark test): MPs did not significantly alter anxiety-like behavior of the zebrafish. Social/shoaling behavior: MPs altered social behavior significantly, leading to tighter shoaling patterns.	[64]
	PS/Spherical beads/2 μm diameter/0.44 mg/L (~108 items/L)	Locomotion behavior: Average swimming velocity (ASV), duration of high mobility (DHM), frequency of high mobility (FHM), duration of thigmotaxis (DTH).	7 d	Results: No locomotion alteration. No change in thigmotaxis or high mobility metrics. No change in post-stimulation escape response. While there is a significant increase in shoaling duration. While it enhances the shoaling behavior, it did not induce hyperactivity or hypoactivity or alter startle performance.	[66]
	PE/Not disclosed/60 mg/L	Behavioral endpoints: School cohesion, school depth, distance from predator (anti-predator response deficit inferred).	10 d	Results: Direct exposure to PE MPs at 60 mg/L for 10 days did not significantly alter: Latency to reach the top, time spent in the top zone, anxiety index in open field test, swimming speed, or total distance traveled. Additionally, no significant increase in shoal cohesion when predator was present, impaired defensive aggregation response, and reduced prey–predatory distance compared to control.	[78]
	PGA/~1 μm in diameter/1 mg/L & 100 mg/L	Behavior: anxiety-like behavior + cognitive/visual preference behaviors (novel tank and color preference experiments).	28 d	After zebrafish exposed to 1 mg/L and 100 mg/L of PGA microplastics for 28 days, some clear changes showed up in their behavior. First, in the novel tank test, the fish started sticking to the bottom more as the concentration went up. Their movement became really limited to the bottom zone. Significant decreased in: Time spent in the top zone; Distance traveled in the top zone; The ratio of time spent top versus bottom; Number of times they entered the top zone. Interestingly, their average swimming speed did not change much. For cognitive effects, researchers used a color preference test with a four-arm maze. Normally, the fish prefer colors in this order: Blue, then no color, then green, red, and yellow last. After exposure: The high concentration group spent more time in the blue and no-color areas. Their color preference shifted compared to the control group. The low concentration group did not show much change in color preference.	[47]
	PS/Spheres/0.1 μm diameter/0, 0.1, 1, 10, 50, 100 mg/L	Locomotion: Moving distance, swimming speed.	96 h	Swimming pace through the trial did not change reliably when it compares to the control. The pattern of behavior seen in PS-MP-treated subjects matched levels observed in animals getting no treatment. At 1 mg/L, polystyrene MPs it showed no obvious change in fish behavior after 96 h. Movement patterns stayed the same regardless of exposure.	[48]
Embryos → Larvae	PS/Spherical/1 µm in diameter/0.1, 1, and 10 mg/L	Swimming velocity, mobility ratio, avoidance response.	21 d	Swimming velocity: No significant effect in MP exposure. Mobility ratio: No significant effect. Avoidance response: No significant differences relative to the control for MPs alone.	[86]
	PS/Spherical/1 µm diameter/100 µg/L and 1000 µg/L	Swimming distance, swimming speed, and Dark-light avoidance behavior	4 hpf → 96–120 hpf	Swimming speed dropped by 3.5% when fish were exposed to 1000 μg/L in dark water after 120 hpf. That level reduced movement more than any other. Swimming speed dropped slightly—1.5 plus or minus 0.3 mm per second compared to 1.5 plus or minus 0.3 in healthy animals; that difference turned out statistically significant, p equals 0.03. At 1000 micrograms per liter, overall movement shrunk, cutting the total distance by roughly 3.2 percent. A shift appeared with 15.3 ± 2.8 cm measured against 15.8 ± 2.7 cm baseline values—difference found significant at p = 0.021. Exposure to 100 μg/L showed no clear impact during dark swimming trials. Light and dark cycles switched every few days in a test lasting around five days at 120 hpf. Light period: Swimming length and speed showed no clear changes across the board—whether at 100 or 1000 μg/L. Dark period: At 100 μg/L, fish swam shorter distances—by 4.6% fewer beats. Swimming slowed down by 4.9% at 100 μg/L. A shift appeared—movement slowed in one condition compared to the other. Speed dropped by about 1.41 to 1.48 mm per second. That difference was highly significant, showing up clearly beyond doubt. The p value hit a near-zero mark after testing. At 1000 μg/L, animals swam 2.8 percent slower. Speed rose slightly in treatment versus control (1.44 ± 0.24 mm/s vs. 1.48 ± 0.23 mm/s; p = 0.0065).	[99]
	PGA, PLA, PBS, PHA, PBAT/No data/1 mg/L and 100 mg/L for each polymer	Behavioral: Thigmotaxis at 3 dpf (edge preference); light–dark test at 5 dpf: max speed, total distance, movement count in light vs. dark.	3, 6, 10, 24, and 96 hpf	Thigmotaxis behavior in 3-day-old larvae: Larvae exposed to strong concentrations—like 100 mg/L—of PGA, PLA, PBS, and PBAT showed a clearer tendency toward edge avoidance. This shift toward thigmotaxis became more pronounced, suggesting heightened anxiety behavior. Even at 100 mg/L, PHA did not cause much thigmotactic response. At just 1 mg/L, there was barely any change in thigmotaxis worth noting. Larvae at five days old responded to changes in light and dark. During shifts in illumination, their actions were observed. Light at 80 lux levels: Every single one of the five natural breakdown microplastics—made from PGA, PLA, PBS, PHA, PBAT—dissolved more easily when tested at both light and heavy doses. Their ability to break down happened clearly across every sample exposure. Dark conditions (0.1×): At 100 mg/L PBS and 1 mg/L PBAT caused slower movement, lower overall travel, and fewer shifts in position. At 100 mg/L PHA cut down movement speed and overall motion total. Not every option lowered dark-phase movement—some changes did not affect it significantly. Light–dark responsiveness: Beyond 100 mg/L PBS plus 1 mg/L PBAT, movement patterns stayed largely unchanged across light-to-dark shifts. These conditions weakened the body’s reliance on visual cues when responding to sudden changes. Light-driven reactivity simply fell short under such exposure. Other groups still showed clear differences in movement between light and dark periods.	[62]
Larvae	PS & PVC/Spherical/PS (Spherical ~7.0 µm mean size) PVC (Spherical ~3.8 µm mean size)/Both MPS 20 mg L−1	Immobility duration, mean swimming velocity, turn angle (locomotor behavior)	10 d	Locomotor activity: PS & PVC MPs significantly suppressed swimming activity compared to control. Immobility duration: Immobility duration significantly increased in larvae and immobility in PVC group showed U-shaped trajectory, indicating high immobility observed under PVC-MP exposure. Mean velocity and turn angle: Both PS & PVC-MPs reduced the mean swimming velocity of zebrafish. The turn angle was altered in a concentration-dependent manner.	[57]
	Virgin & Photo-aged PS/Virgin: 10 μm Photo-aged: 6.5 μm/0.1–100 μg/L for both V-PS and P-PS.	Behavior: Locomotor activity (reduced locomotion).	1 hfp to 120 hpf	Locomotor activity: After 120 h of development, young zebrafish moved less when exposed to 100 μg/L of virgin PS-MPs than those in clean water. Their ability to swim declined noticeably under that condition. Swimming changes showed up when larvae were exposed to photo-aged PS-MPs at 1–100 μg/L levels. Speed shifted in response to those doses. P-PS given at 10 and 100 μg/L caused fish to swim slower, with lower average speeds than in the control and V-PS groups (p < 0.05). Light–dark response: Most changes happened while the lights were off. The P-PS group showed clearer shifts than others. Correlation with neurochemical biomarkers measures of behavior—like how fast zebrafish swam—tended to drop as brain chemicals such as 5-HT, GABA, and ACh went down. What also followed a similar pattern was the activity of enzymes like AChE and ChE, which moved in step with those neurotransmitters but in opposite directions.	[47]

Abbreviations: PS—polystyrene; PP—polypropylene; PE—polyethylene; PES—polyester; PVC—polyvinyl chloride; PGA—polyglycolic acid; PBAT—polybutylene adipate terephthalate; PLA—polylactic acid; PHA—polyhydroxyalkanoate; PBS—polybutylene succinate; PET—polyethylene terephthalate; PA—polyamide; MPs—microplastics; hpf—hours post-fertilization; dpf—days post-fertilization; h—hours; d—days; w—weeks; →—progression.

Table 3. Behavioral effects of pristine/virgin/unspecifies and UV/photo-aging in MP exposure.

Life Stage	Plastic Characterization (Type/Shape/Size/Concentrations)	Endpoints	Exposure Time	Effects	Ref.
Adult	PS/Irregular fragments/~400 µm/0.5 mg/L	Behavioral parameters: Latency to first MP capture, capture frequency, spitting frequency, swallowing ratio (%), feeding duration, time in feeding zone, swimming activity, speed, total distance moved.	10 m per trial	Latency to first capture: Most zebrafish started MP capture within ~10 s of adding MPs. Shy zebrafish showed longer latency than bold zebrafish (not significant). Capture frequency: Bold zebrafish captured MPs significantly more often than shy zebrafish on exposure days (p < 0.05). Spitting behavior: Zebrafish often spat MPs out after capture. About 30–47% of total capture events happened in the first minute. Spitting frequency was positively correlated with capture frequency. Swallowing ratio: About 40–60% of captured MPs were swallowed. Bold fish had higher effective exposure because of higher capture frequency. Swimming activity during feeding: Zebrafish showed immediate increase in activity after MP addition. Bold zebrafish showed significantly higher levels of feeding activity (activity %, total distance, average speed) than shy zebrafish on days 2–3 (p < 0.05). Bold zebrafish spent significantly more time in feeding areas (upper water layer). Ingestion/egestion behavior: Bold zebrafish ingested more MPs than shy zebrafish when exposed to MPs only (significant on day 2). Very few MPs were left in intestines after 3 days, suggesting efficient egestion.	[96]
	Propriety polymer (composition undisclosed/Spherical/1–5 µm/2 mg/L	Behavior: Locomotor activity (Open Field Test), anxiety (Light-Dark Test), and social.	30 d	Locomotor activity (open field test): Mean speed: Markedly reduced in MP group (584 ± 33.5 cm/min) compared to control (696 ± 27.1 cm/min) (p < 0.01). Total distance moved: Significantly reduced in MP group (2863.4 ± 1.8 cm) compared to control (3445.8 ± 234.4 cm) (p = 0.0395). Absolute turn angle: Not significantly different between MP group and control. Time inactive: Significantly increased in MP group (10.4 ± 0.3%) compared to control (3.0 ± 0.3%) (p < 0.0001). Time in center zone: no significant difference between MP group and control. Anxiety-like behavior (light–dark test): MPs did not significantly alter anxiety-like behavior of the zebrafish. Social/shoaling behavior: MPs altered social behavior significantly, leading to tighter shoaling patterns.	[64]
	PS/Spherical beads/2 μm diameter/0.44 mg/L (~108 items/L)	Locomotion behavior: Average swimming velocity (ASV), duration of high mobility (DHM), frequency of high mobility (FHM), duration of thigmotaxis (DTH).	7 d	Results: No locomotion alteration. No change in thigmotaxis or high mobility metrics. No change in post-stimulation escape response. While there is a significant increase in shoaling duration. While it enhances the shoaling behavior, it did not induce hyperactivity or hypoactivity or alter startle performance.	[66]
	PE/Not disclosed/60 mg/L	Behavioral endpoints: School cohesion, school depth, distance from predator (anti-predator response deficit inferred).	10 d	Results: Direct exposure to PE MPs at 60 mg/L for 10 days did not significantly alter: Latency to reach the top, time spent in the top zone, anxiety index in open field test, swimming speed, or total distance traveled. Additionally, no significant increase in shoal cohesion when predator was present, impaired defensive aggregation response, and reduced prey–predatory distance compared to control.	[78]
	PGA/~1 μm in diameter/1 mg/L & 100 mg/L	Behavior: anxiety-like behavior + cognitive/visual preference behaviors (novel tank and color preference experiments).	28 d	After zebrafish exposed to 1 mg/L and 100 mg/L of PGA microplastics for 28 days, some clear changes showed up in their behavior. First, in the novel tank test, the fish started sticking to the bottom more as the concentration went up. Their movement became really limited to the bottom zone. Significant decreased in: Time spent in the top zone; Distance traveled in the top zone; The ratio of time spent top versus bottom; Number of times they entered the top zone. Interestingly, their average swimming speed did not change much. For cognitive effects, researchers used a color preference test with a four-arm maze. Normally, the fish prefer colors in this order: Blue, then no color, then green, red, and yellow last. After exposure: The high concentration group spent more time in the blue and no-color areas. Their color preference shifted compared to the control group. The low concentration group did not show much change in color preference.	[47]
	PS/Spheres/0.1 μm diameter/0, 0.1, 1, 10, 50, 100 mg/L	Locomotion: Moving distance, swimming speed.	96 h	Swimming pace through the trial did not change reliably when it compares to the control. The pattern of behavior seen in PS-MP-treated subjects matched levels observed in animals getting no treatment. At 1 mg/L, polystyrene MPs it showed no obvious change in fish behavior after 96 h. Movement patterns stayed the same regardless of exposure.	[48]
Embryos → Larvae	PS/Spherical/1 µm in diameter/0.1, 1, and 10 mg/L	Swimming velocity, mobility ratio, avoidance response.	21 d	Swimming velocity: No significant effect in MP exposure. Mobility ratio: No significant effect. Avoidance response: No significant differences relative to the control for MPs alone.	[86]
	PS/Spherical/1 µm diameter/100 µg/L and 1000 µg/L	Swimming distance, swimming speed, and Dark-light avoidance behavior	4 hpf → 96–120 hpf	Swimming speed dropped by 3.5% when fish were exposed to 1000 μg/L in dark water after 120 hpf. That level reduced movement more than any other. Swimming speed dropped slightly—1.5 plus or minus 0.3 mm per second compared to 1.5 plus or minus 0.3 in healthy animals; that difference turned out statistically significant, p equals 0.03. At 1000 micrograms per liter, overall movement shrunk, cutting the total distance by roughly 3.2 percent. A shift appeared with 15.3 ± 2.8 cm measured against 15.8 ± 2.7 cm baseline values—difference found significant at p = 0.021. Exposure to 100 μg/L showed no clear impact during dark swimming trials. Light and dark cycles switched every few days in a test lasting around five days at 120 hpf. Light period: Swimming length and speed showed no clear changes across the board—whether at 100 or 1000 μg/L. Dark period: At 100 μg/L, fish swam shorter distances—by 4.6% fewer beats. Swimming slowed down by 4.9% at 100 μg/L. A shift appeared—movement slowed in one condition compared to the other. Speed dropped by about 1.41 to 1.48 mm per second. That difference was highly significant, showing up clearly beyond doubt. The p value hit a near-zero mark after testing. At 1000 μg/L, animals swam 2.8 percent slower. Speed rose slightly in treatment versus control (1.44 ± 0.24 mm/s vs. 1.48 ± 0.23 mm/s; p = 0.0065).	[99]
	PGA, PLA, PBS, PHA, PBAT/No data/1 mg/L and 100 mg/L for each polymer	Behavioral: Thigmotaxis at 3 dpf (edge preference); light–dark test at 5 dpf: max speed, total distance, movement count in light vs. dark.	3, 6, 10, 24, and 96 hpf	Thigmotaxis behavior in 3-day-old larvae: Larvae exposed to strong concentrations—like 100 mg/L—of PGA, PLA, PBS, and PBAT showed a clearer tendency toward edge avoidance. This shift toward thigmotaxis became more pronounced, suggesting heightened anxiety behavior. Even at 100 mg/L, PHA did not cause much thigmotactic response. At just 1 mg/L, there was barely any change in thigmotaxis worth noting. Larvae at five days old responded to changes in light and dark. During shifts in illumination, their actions were observed. Light at 80 lux levels: Every single one of the five natural breakdown microplastics—made from PGA, PLA, PBS, PHA, PBAT—dissolved more easily when tested at both light and heavy doses. Their ability to break down happened clearly across every sample exposure. Dark conditions (0.1×): At 100 mg/L PBS and 1 mg/L PBAT caused slower movement, lower overall travel, and fewer shifts in position. At 100 mg/L PHA cut down movement speed and overall motion total. Not every option lowered dark-phase movement—some changes did not affect it significantly. Light–dark responsiveness: Beyond 100 mg/L PBS plus 1 mg/L PBAT, movement patterns stayed largely unchanged across light-to-dark shifts. These conditions weakened the body’s reliance on visual cues when responding to sudden changes. Light-driven reactivity simply fell short under such exposure. Other groups still showed clear differences in movement between light and dark periods.	[62]
Larvae	PS & PVC/Spherical/PS (Spherical ~7.0 µm mean size) PVC (Spherical ~3.8 µm mean size)/Both MPS 20 mg L−1	Immobility duration, mean swimming velocity, turn angle (locomotor behavior)	10 d	Locomotor activity: PS & PVC MPs significantly suppressed swimming activity compared to control. Immobility duration: Immobility duration significantly increased in larvae and immobility in PVC group showed U-shaped trajectory, indicating high immobility observed under PVC-MP exposure. Mean velocity and turn angle: Both PS & PVC-MPs reduced the mean swimming velocity of zebrafish. The turn angle was altered in a concentration-dependent manner.	[57]
	Virgin & Photo-aged PS/Virgin: 10 μm Photo-aged: 6.5 μm/0.1–100 μg/L for both V-PS and P-PS.	Behavior: Locomotor activity (reduced locomotion).	1 hfp to 120 hpf	Locomotor activity: After 120 h of development, young zebrafish moved less when exposed to 100 μg/L of virgin PS-MPs than those in clean water. Their ability to swim declined noticeably under that condition. Swimming changes showed up when larvae were exposed to photo-aged PS-MPs at 1–100 μg/L levels. Speed shifted in response to those doses. P-PS given at 10 and 100 μg/L caused fish to swim slower, with lower average speeds than in the control and V-PS groups (p < 0.05). Light–dark response: Most changes happened while the lights were off. The P-PS group showed clearer shifts than others. Correlation with neurochemical biomarkers measures of behavior—like how fast zebrafish swam—tended to drop as brain chemicals such as 5-HT, GABA, and ACh went down. What also followed a similar pattern was the activity of enzymes like AChE and ChE, which moved in step with those neurotransmitters but in opposite directions.	[47]

Abbreviations: PS—polystyrene; PP—polypropylene; PE—polyethylene; PES—polyester; PVC—polyvinyl chloride; PGA—polyglycolic acid; PBAT—polybutylene adipate terephthalate; PLA—polylactic acid; PHA—polyhydroxyalkanoate; PBS—polybutylene succinate; PET—polyethylene terephthalate; PA—polyamide; MPs—microplastics; hpf—hours post-fertilization; dpf—days post-fertilization; h—hours; d—days; w—weeks; →—progression.

6. Summary and Future Perspectives

The studies included in this systematic review consistently demonstrate that MP toxicity in zebrafish is strongly influenced by polymer type, particle size, concentration, exposure duration, and weathering status. However, considerable methodological heterogeneity among studies limits direct comparison of toxicological outcomes. Future research should therefore prioritize methodological standardization to improve reproducibility and ecological relevance.

• Future studies should prioritize standardized reporting of microplastic physicochemical properties, including polymer identity, particle size distribution, morphology, aging status, surface characteristics, and concentration metrics. The reviewed studies used diverse exposure designs and reporting approaches, making direct comparisons difficult and limiting quantitative synthesis. Establishing harmonized experimental protocols would improve reproducibility and facilitate cross-study comparisons.
• Although advanced analytical approaches, including imaging technologies, omics analyses, and adverse outcome pathway (AOP) frameworks, may contribute to future mechanistic investigations, relatively few of the studies included in this review employed these approaches. In addition, these advanced methodologies are presented only as potential research opportunities rather than evidence-based conclusions arising from the present systematic review.
• Artificial intelligence and machine learning technologies are emerging as promising tools in MP ecotoxicology research. AI image analysis is seen as having the potential to enhance MP detection, identification, and quantification in environmental samples and biological tissues. Machine learning can also be applied to analyze large datasets from multi-omics approaches to identify predictive toxicity pathways and biomarker networks. In zebrafish behavioral assays, AI video tracking systems are being increasingly applied to quantify locomotor activity, anxiety-like behavior, and social behavior with greater accuracy and without subjective bias.

The collated experimental evidence confirmed that MPs are biologically active stressors in zebrafish, which could trigger coordinated dysfunctions at developmental, physiological, and neurobehavioral levels. The results emphasize the importance of standardized experimental approaches to ensure ecological relevance and risk assessment. In the future direction of using zebrafish as a model for conducting MP toxicity studies, it is recommended that there be an increased focus on advancing the knowledge in this field in terms of mechanistic understanding, ecological relevance, and translational value. Future studies should aim to expand beyond the current single-generation design and include multi-generation studies to assess transgenerational toxicity. Moreover, the use of “omics” technologies such as transcriptomics, proteomics, metabolomics, and epigenomics is essential for advancing our knowledge in the field of microplastic toxicity. Furthermore, standardization of the parameters used in conducting microplastic toxicity studies is essential for increasing the reproducibility and comparison of the data generated in this field. Future studies should also aim to include environmentally relevant microplastics in the studies. This can be achieved by using aged microplastics and those with biofilm formation. Also, future studies should also include co-exposure studies with other environmental contaminants. Moreover, the use of imaging technologies will be useful in precisely assessing the accumulation of microplastics in the tissues. Additionally, further neurobehavioral studies, including circadian rhythm studies and cognitive function tests, are necessary to more fully understand the effects on the nervous system. Microbe–gut interactions involving MPs should also be explored to understand this relationship. The development of high-throughput screening tools to automate testing procedures for toxicity studies is also important to increase efficiency in testing. The integration of zebrafish-derived data into adverse outcome pathways/ecological risk assessment frameworks is important to fully utilize this model in assessing the overall effects of microplastic pollution.

Finally, based on the findings of this systematic review, future zebrafish studies should focus on improving methodological consistency by adopting standardized microplastic characterization, environmentally relevant exposure scenarios, validated weathering procedures, and harmonized biological endpoints across developmental, physiological, and neurobehavioral assessments. These improvements will strengthen evidence synthesis, facilitate comparison among studies, and improve ecological risk assessment of microplastics.