Behavioural impact of microplastics on zebrafish development

Behavioural impact of microplastics on zebrafish development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Behavioural impact of microplastics on zebrafish development Andrea Cázares-Morales, Nallely Magaña-Montiel, Liliana Pardo-López, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8398009/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 15 You are reading this latest preprint version Abstract Microplastic (MP) pollution in aquatic environments is ubiquitous and characterized by particles of highly irregular shapes. Yet, most laboratory studies examining MP impacts on aquatic species rely on pristine polymer spheres, which poorly reflect the diversity and complexity of environmentally derived MPs. In this study, we assessed the developmental effects of environmentally relevant, household-derived, irregular microplastic fragments, on zebrafish. Fragments of synthetised MPs used in this study displayed heterogeneous sizes and jagged shapes, similar to environmental MPs fragments. Acute exposure to all MP types did not induce embryonic lethality or gross malformations but did result in significant sublethal toxicity: exposed larvae showed reduced touch-evoked escape responses, consistent with a pronounced loss or damage of lateral line neuromasts. To further characterize the underlying sensory impairment, we examined neuromast structure and function, which revealed mechanical disruption, kinocilia fusion, and reduced mitochondrial activity. Our findings emphasize that physical and physicochemical interactions associated with fragment morphology and polymer type drive neurosensory toxicity than particle size alone. This work highlights that acute MP exposure disrupts key sensory behaviours and structures critical for ecological fitness. Overall, these results support the need for increased regulatory and scientific attention to behavioural and sensory endpoints in microplastic risk assessments. Biological sciences/Ecology Earth and environmental sciences/Ecology Earth and environmental sciences/Environmental sciences Biological sciences/Neuroscience Biological sciences/Zoology microplastics zebrafish neuromasts toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Plastic production has increased exponentially since its widespread use in the 1950s, with more than half of all plastic ever produced being generated between 2000 and 2020 alone [ 1 ]. As the use and disposal of plastic escalates, and because of its slow degradation rate, the persistent accumulation of plastic debris is observed in both aquatic and terrestrial environments [ 2 ]. Moreover, particularly in marine ecosystems, larger plastic items are subject to photooxidation and mechanical degradation, fragmenting into microplastics [ 3 ]. Microplastics (MPs) are particles smaller than 5 mm and are highly heterogeneous, varying in polymer composition, size, colour, and shape [ 2 ]. These features influence their biological activity and environmental fate [ 1 ]. Although MPs are generally considered non-lethal at environmental concentrations, growing evidence links MP exposure to a broad spectrum of adverse biological effects, including neurodevelopmental and immune dysfunction, endocrine disruption, gastrointestinal disturbances, metabolic and cardiovascular impairments, oxidative stress, and microbiota dysbiosis [ 4 ]. Notably, MPs have recently been detected in human placental tissue, meconium, and breast milk, raising concerns about the potential adverse effects of MP exposure during early development [ 5 , 6 ]. Most experimental studies investigating microplastic toxicity have relied primarily on pristine, laboratory-generated particles—typically uniform spheres—composed of a single polymer [ 7 , 8 ]. This approach contrasts sharply with environmental reality, where microplastics detected in air, water, and food are predominantly heterogeneous fibers and fragments [ 1 ]. The exclusive use of pristine spheres and limited polymer types in toxicological studies can underestimate or misrepresent the true ecological risks posed by microplastics. It has been suggested that toxicity increases with decreased particle size and is further amplified by specific shapes. Fibers are often more hazardous than fragments, which in turn are more toxic than spheres [ 9 ]. Fibers and fragments, often weathered and chemically altered, exhibit greater toxicity and are more environmentally relevant, reflecting the diversity and complexity of real-world exposures [ 9 – 11 ]. Additionally, toxicological studies have disproportionately focused on a few MPs, overlooking the diversity and weathering found in environmental samples [ 1 ]. This approach can have profound consequences as physicochemical properties, such as polymer type, shape, size, colour, surface chemistry, molecular structure, density, and environmental aging, all influence toxicological outcomes, affecting MPs' bioactivity, sorption capacities, and interactions with organisms [ 4 , 10 , 11 ]. The assessment of a broader range of microplastic shapes and chemical compositions is therefore essential to provide meaningful insights into the health of aquatic organisms and to improve environmental risk assessment studies. Zebrafish ( Danio rerio ) embryos and larvae have emerged as a powerful tool in MP research, due to their rapid external development, optical transparency, and high genetic and physiological homology to humans [ 12 , 13 ]. Zebrafish produce hundreds of offspring per clutch, enabling large sample sizes and robust statistical analyses [ 14 ]. Their direct exposure to waterborne MPs mirrors realistic environmental conditions and enables sensitive detection of effects across developmental, behavioural, and molecular endpoints [ 15 ]. Current scientific reports reveal a spectrum of MP-related effects in zebrafish, from early hatching or reduced survival [ 16 ], developmental changes in morphometric parameters [ 11 ], and cardiotoxicity, to downregulation of nervous system and metabolic gene pathways [ 17 ]. Behavioural studies describe seizure-like activity, loss of swimming competence, and altered neurochemical profiles [ 8 ]. MPs have also been implicated in disrupting the microbiome [ 18 ], compromising visual system integrity [ 19 ], and causing genotoxicity [ 20 ]. Importantly, some studies report adverse outcomes even at the lowest environmentally relevant concentrations [ 11 ], while others note little influence on growth or hatching, detecting only changes in gene expression or subtle physiological alterations [ 17 ]. Nevertheless, most studies performed in zebrafish have mainly used polystyrene (PS) spheres [ 4 , 7 ]. In the present study, we systematically produced and characterized a MPs representing those found in environmental samples, such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC), polymers of everyday household items [ 1 ]. Zebrafish embryos were exposed to these MPs from 4 to 120 hours post-fertilization, with assessments of survival, hatching, touch-evoked escape behaviour, and motility. While general swimming capacity was unaffected, exposure impaired the touch-evoked response, prompting focused investigation on neuromast integrity and cell death. The neuromasts of zebrafish are specialized sensory organs that form part of the lateral line system, enabling the detection of water movements and vibrations in the surrounding environment. Neuromasts develop during embryogenesis from migrating primordia that deposit clusters of cells at stereotypical positions along the head and body surface [ 12 ]. Each neuromast consists of mechanosensory hair cells (analogous to those in the mammalian inner ear) interspersed with supporting and mantle cells, all covered by a gelatinous cupula that transmits mechanical stimuli to the hair cells underneath [ 21 , 22 ]. The precise arrangement and continual regeneration of hair cells within each neuromast provides zebrafish with robust and adaptable flow-sensing capabilities, underlying essential behaviours such as rheotaxis, prey detection, predator avoidance, and schooling [ 22 ]. The accessibility and regenerative capacity of lateral line neuromasts make them an invaluable model for studying hair cell biology, sensory organ development, and environmental toxicology. By mapping hazardous outcomes across developmental and sensory endpoints and comparing polymers, our findings contribute to a more detailed understanding of MP toxicology, essential for assessing ecological risks and guiding policy amid the growing plastic pollution crisis. Identifying and understanding the impacts of MP on early-life development is vital, not only for aquatic organisms but also for informing concerns about potential risks during pregnancy and for future generations. The complex interplay of particle characteristics, environmental modification, and biological response demands that toxicological studies reflect realistic exposures by including diverse, environmentally relevant MP polymers and morphologies. 2. Results 2.1 Characterization of synthesized microplastics The identity of the five synthesized microplastic samples, polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC), was confirmed by FTIR-ATR spectroscopy (Fig. 1 , supplementary Table 1). Characteristic absorption bands for each polymer were compared with reference values from [ 23 ] and spectral matches in the OpenSpecy database [ 24 ]. The PE, PP, PS, and PET samples demonstrated absorption bands that either directly matched or fell within 3 wavenumbers of the literature values (supplementary Table 1). Spectral correlation analysis using OpenSpecy yielded high match scores for PE, PP, PS, and PET to published spectra (r = 0.99, Primpke et al. [ 25 ]). For PVC, differences in the main bands were observed at 1242, 1094, and 611 cm − 1 , although the database still indicated a positive match (r = 0.84) consistent with De Frond et al. [ 26 ]. While all polymers could be matched, the PVC sample showed the lowest spectral agreement, likely due to the presence of additives or plasticizers, as differences in the 1500–600 cm 1 region are often observed when such unbound compounds are present [ 27 ]. Accurate determination of particle size is important as particle dimensions strongly influence environmental fate, biological interactions, and potential toxicity of MPs. Size affects sedimentation, bioavailability, and the likelihood of tissue contact or cellular uptake, making robust size characterization essential for interpreting toxicological outcomes and comparing results across studies. SEM imaging further revealed the surface morphology and size distribution of the synthesized microplastics (Fig. 2 ). The particles exhibited irregular, rough-edged fragments with occasional fibrous structures, consistent with mechanical abrasion used as the preparation method. Median particle length differed by polymer, with PS fragments being the largest (172.87 µm, Q1: 140.22, Q3: 235.46), followed by PVC (171.14 µm, Q1: 123.65, Q3: 245.88), PE (161.72 µm, Q1: 142.25, Q3: 221.93), PP (108.97 µm, Q1: 82.83, Q3: 134.56), and PET (101.62 µm, Q1: 82.83, Q3: 134.56). Pairwise comparisons revealed that PE fragments were significantly longer than PET (p < 0.05), but not different from PP, PS, or PVC. PP fragments were shorter than PS (**p < 0.001) and PVC (*p < 0.01), while no significant differences were detected between PP and PET. PS fragments were longer than PET (***p < 0.0001) and PP, but did not differ from PVC. PET fragments were shorter than PVC (**p < 0.001). These findings indicate clear, polymer-dependent heterogeneity in the size distributions of environmentally relevant MP fragments. Overall, the combined FTIR-ATR and SEM analyses confirm the successful synthesis and accurate identification of the five microplastic types. The chemical and morphological characteristics observed are consistent with the literature. We were able to produce irregular MPs similar to those found in environmental samples, supporting their use in subsequent experiments. 2.2 Microplastic effect in Zebrafish development Zebrafish larvae were exposed to the different types of microplastics. No significant differences were observed in survival, hatching rates, or gross morphology between exposed and control groups (Supplementary Fig. 1). However, a touch-evoked response test showed that microplastics caused a reduction in larvae’s response. We conducted touch-evoked response assays at 72, 96, and 120 hpf (Fig. 3 ). Larvae exposed to microplastics exhibited diminished sensitivity, requiring more tactile stimuli to elicit an escape response. Approximately 80% of control larvae responded to the first stimulus, and the rest responded to a second stimulus at all the times tested (Fig. 3 A and B). At 72hpf, PE-treated larvae reacted similarly to the controls, while the rest of the MP treatments showed that only about half of the larvae responded at the first touch (PP 60.83%; PS 53.85%; PET 59.35% and PVC 49.1%; Fig. 3 A), requiring four to eight stimuli to respond (Fig. 3 B). At 96 hpf, all the MPs showed that at least 50% of the larvae are affected in the touch-evoked response (PE 56.48%; PP 52.06%; PS 52.22%; PET 54.23% and PVC 38.33%). At 96 hpf, we also observed that the larvae required a higher number of stimuli to react and that in some cases, larvae did not react after ten stimuli when the test ended. By 120 hpf, 83.33% of controls reacted to the first stimulus, and PE and PP groups showed no significant difference from controls; nevertheless, an increased number of stimuli was still observed compared to controls (Fig. 3 B), while response rates for PS, PET, and PVC remained below 60% (PE 76.25%; PP 77.96%; PS 50%; PET 58.51% and PVC 58.33%) significantly different from controls. Importantly, once the escape response was initiated, swimming performance and activity duration were comparable to those of controls (Supplementary Fig. 2). These results indicate that, under experimental conditions, microplastic exposure affected the larvae’s sensitivity to tactile stimuli but did not impair neuromotor or motor function. Because altered tactile responsiveness can arise from disruptions in lateral line mechanoreceptors, we next assessed the structural integrity of neuromasts. 2.3 Neuromast characterization To assess the impact of MPs on mechanosensory integrity, the structure of lateral line neuromasts was examined following exposure (Fig. 4 ). SEM analysis at 96 and 120 hpf revealed that neuromasts in untreated controls possessed large, distinct kinocilia. In larvae exposed to PE and PP, kinocilia were absent at 96 hpf, though short kinocilia appeared in the PE and PP groups at 120 hpf. In contrast, larvae treated with PS, PET, or PVC displayed predominantly short kinocilia at 96 hpf, with evidence of possibly kinocilia fusion by 120 hpf. Microplastic particles were frequently observed adhering near or directly to neuromasts (Fig. 4 ). To further characterize the lateral line neuromasts, we used DASPEI staining. The vital dye DASPEI labels hair cell mitochondria in the neuromast and is used to assess their integrity. Our studies demonstrated a significant reduction in neuromast number in the lateral line at 96 hpf for MP-treated groups: while control larvae averaged 8 to 9 neuromasts, exposed groups had only 6 to 7, and counts as low as 4 were observed in some PS or PVC-exposed larvae (Fig. 5 ). By 120 hpf, the PE, PP, and PVC groups showed partial recovery in neuromast number, whereas the PS and PET groups remained lower than controls (Fig. 5 ). At higher magnification (Fig. 6 ), control neuromasts showed hair cells arranged in a convergent, flower-like configuration, with robust and elongated (mitochondria-rich) DASPEI-positive hair cells. At 96 hpf, larvae exposed to MPs displayed weaker fluorescence intensity in neuromast hair cells, indicative of reduced hair cell density or integrity. By 120 hpf, increased DASPEI staining was noted in PE- and PVC-treated larvae compared to 96 hpf, although mitochondria appeared shorter than the typical elongated architecture observed under normal conditions (Fig. 6 ). Fluorescence was generally weaker with PS and PET exposure than in controls. Incomplete or patchy staining was particularly pronounced at 96 hpf for all MP groups, and only partial recovery of hair cell structure was observed by 120 hpf of PE and PVC, with the mitochondrial network less distinct than in controls (Fig. 6 ). To assess the integrity of the neuromast hair cells, we conducted dual staining with acridine orange to detect cell death and Bodipy to visualize the membranes of the neuromast cells at 96 and 120 hpf. The staining revealed no significant differences in hair cell death within neuromasts between control and MP-exposed larvae (Fig. 7 ). Further staining with Bodipy enabled visualization of both hair cells and supporting cells within the neuromast, with no evident architectural alterations between treated and control groups (Fig. 7 ). Notably, although acridine orange staining revealed no significant differences in cell death between control and microplastic-exposed groups, collectively, exposure to microplastics during early zebrafish development disrupts the function of lateral line neuromasts. Marked alterations were consistently observed in neuromast cilia morphology and mitochondrial network that alter the function in the exposed larvae, consistent with the lack of touch-evoke response in the treated zebrafish larvae. Changes in kinocilia morphology and reduced numbers of DASPEI-positive hair cells suggest impaired mechanosensory function. The persistence of these defects, even with some recovery at later stages, highlights the sensitivity of neuromasts to microplastic-induced damage and underscores the risk to sensory development and function in aquatic organisms exposed to environmentally relevant levels of microplastics. Taken together, these findings indicate that acute exposure to environmentally relevant, irregular microplastic fragments causes significant sublethal effects in developing zebrafish. Larvae showed reduced touch-evoked escape responses and clear disruption of lateral line neuromasts, including mechanical damage, kinocilia fusion, and decreased mitochondrial activity. All tested polymers induced damaged neuromast integrity, with PS, PET, and PVC causing the strongest effects. These results suggest that material properties and fragment shape are key factors in neurosensory toxicity. 3. Discussion Microplastic pollution in aquatic environments is associated with a broad spectrum of biological effects in zebrafish, including neurodevelopmental toxicity [ 8 , 17 ], gastrointestinal disturbance [ 28 ], microbiota dysbiosis [ 18 ], cardiac dysfunctions [ 16 , 29 ], and metabolic imbalances [ 2 , 30 ]. While these adverse outcomes are well documented, the mechanisms underlying sublethal neurotoxicity and physical damage, especially at early developmental stages, remain poorly understood. In contrast to most studies that use pristine, single polymers, our work used irregular MPs generated from common household materials, capturing the heterogeneity of polymer types and surface features encountered in real-world conditions. Although the exposure concentration (1 mg/mL) may seem high compared to most levels reported in environmental matrices, it was selected to represent a conservative worst-case scenario and to ensure detection of polymer-specific effects that could otherwise be absent at lower doses. Nevertheless, some reports indicate that MPs can reach or exceed the concentration applied here in highly impacted contexts; MP concentrations of 1.8 mg/mL have been reported in wastewater from a plastic recycling facility [ 31 ], demonstrating the potential for extremely elevated local contamination levels; MP surface loadings of 3.63 mg/cm² have been reported along the Canary Islands coastline [ 32 ], indicating substantial accumulation of MPs in coastal depositional zones.; and MP concentrations in human liver and brain have been reported to increase significantly between 2016 and 2024, with brain levels in 2024 reaching 4.917 mg/g and higher burdens observed in individuals with dementia than in healthy controls [ 33 ]. Taken together, these data suggest that although our exposure concentration could exceed typical environmental levels, it falls within the range observed in heavily contaminated systems and in human tissues, supporting the toxicological and hazard-oriented relevance of the dose used in our experiments. The zebrafish lateral line is an increasingly used model for investigating ototoxicity and is a suitable alternative to other vertebrate models, as it reproduces ototoxic responses similar to those observed in humans exposed to various compounds [ 34 ]. Neuromasts are the lateral line sensory organs that detect water movement and enable behaviors like predator avoidance and schooling [ 22 ]. Each neuromast contains mechanosensory hair cells whose stereocilia and a kinocilium deflect with water flow, opening the mechanoelectrical transduction (MET) channels, depolarizing the cell, and triggering calcium-dependent neurotransmitter release onto afferent neurons [ 21 , 35 ]. Because hair cells have high energetic demands, they are particularly vulnerable to mitochondrial disruption. In neuromast hair cells, mitochondria occupy a substantial portion of the cell and form an extensive, interconnected network [ 35 ]. Proper mechanotransduction is essential for the development of this specialized mitochondrial architecture, while ongoing synaptic transmission promotes the growth and specific localization of large mitochondria. Disruption of mechanotransduction or synaptic activity can alter mitochondrial structure and compromise hair cell function [ 35 ]. The impaired touch-evoked escape responses observed in our study suggest that household-derived MPs compromise mechanotransduction in zebrafish larvae. The reduced DASPEI labeling may reflect either inhibition of MET channel function or degeneration of hair bundle cilia. Exposure to MPs resulted in striking morphological alterations kinocilia appeared fused or tangled and the number and intensity of DASPEI-positive hair cells were reduced, particularly after PS and PET treatment. Our results are consistent with previous findings that large MP fragments and high doses cause agglomeration of hair cell bundles [ 36 ]. These results are also consistent with impaired MET channel function, as fused hair bundles likely hinder channel opening, thereby restricting dye entry and decreasing fluorescence [ 36 ] causing a decline at the cation influx necessary to induce a signal. Neuromasts contain a mosaic of both mature and young hair cells [ 35 ], and MP exposure may disrupt this homeostatic renewal, compounding sensory deficits over developmental time. In MP‑treated neuromasts, we observed fragmented, abnormally short mitochondria, whereas control larvae displayed the typical architecture with numerous large basal mitochondria supporting synaptic function and smaller apical mitochondria; damage to this network is associated with reduced membrane potential, decreased ATP production, and increased susceptibility to apoptosis [ 10 , 35 ]. Notably, in some neuromasts, the cilia that were absent at earlier time points appeared again, consistent with the ongoing renewal and regeneration of hair cells in the lateral line. Ototoxic compounds provide a useful framework for interpreting the neuromast damage observed in MP‑exposed larvae, as many of the structural and functional alterations resemble classic hair‑cell ototoxicity patterns described in the zebrafish lateral line model. Metals such as copper, manganese, and cobalt rapidly impair mechanotransduction and neuromast function, either by disrupting stereocilia organization and blocking MET channel, or by decreasing afferent neuron sensitivity, with high copper doses even preventing regeneration [ 37 , 38 ]. Similarly, antibiotics (neomycin) and antitumoral drugs (cisplatin) induce hair‑cell death through combined disruption of Ca²⁺ homeostasis in the endoplasmic reticulum–mitochondria dynamic, mitochondrial overload, and excessive ROS generation, ultimately leading to loss of membrane potential, activation of apoptotic cascades [ 39 , 40 ]. Within this ototoxic context, the reduced DASPEI staining, impaired touch‑evoked responses, and hair‑bundle abnormalities caused by household‑derived MPs align with a broader paradigm in which diverse xenobiotics converge on a common target, mechanotransduction channels, mitochondrial integrity, and oxidative balance to compromise neuromast function and sensorimotor performance. Oxidative stress is among the most frequently reported adverse effects associated with exposure to MPs [ 1 , 41 ]. During MP breakdown, free radicals are generated within the polymer chains and can react with oxygen to form additional reactive species. The chemical structure and composition of each polymer play a crucial role in determining its susceptibility to free radical formation during weathering and degradation in the marine environment [ 3 ]. Mitochondria-rich hair cells are particularly vulnerable to oxidative stress and high metabolic demands [ 35 , 42 ]. Thus, the observed loss of mechanosensory function and morphologic integrity in lateral line neuromasts is likely mediated by a combination of mechanical abrasion, impaired ion transport, mitochondrial dysfunction, and oxidative damage, culminating in impaired sensorimotor and behavioral capacity in exposed zebrafish larvae. Hydrogen peroxide exposure has been reported to damage neuromasts by inducing kinociliary abnormalities, promoting hair cell detachment from the neuromast rosette, and ultimately leading to hair cell death [ 42 ]. Such oxidative stress disrupts the structural and functional integrity of hair cells, diminishing their ability to detect mechanical stimuli and compromising overall sensory performance. Although oxidative stress was not directly measured in this study, its contribution to the observed effects cannot be ruled out. Other studies in marine, freshwater, and terrestrial organisms, including fish, crustaceans, mollusks, and rotifers, reported that PE, PP, PS, PET, and PVC can induce both oxidative stress and neurotoxicity [ 43 ]. The evidence indicates that microplastics primarily induce neuromast damage through mechanical abrasion of the epithelial surface, as the relatively large fragment size used prevents cellular internalization. The results are best explained by physical contact and surface injury rather than internalized chemical mechanisms. Notably, PS, PET, and PVC were associated with the most pronounced neuromast and behavioral impairments. This pattern does not strictly follow the conventional idea that smaller MPs are always more toxic; PS in our study represented the largest particle size yet induced severe effects, while PVC was among the smallest and caused marked toxicity. Fragment shape did not vary significantly across polymer types, pointing to physicochemical differences as key explanatory factors. While additional factors such as density, surface hardness, and hydrophobicity are established in the literature as important determinants of microplastic toxicity, their impacts are inferred here based on the known properties of each polymer and are not directly measured. Therefore, our findings highlight that even among irregular fragments of similar shape, polymer-specific composition, and resulting differences in particle–tissue interactions play a central role in the observed patterns of neuromast damage and behavioral impairment. The enhanced impact of PS, PET, and PVC in our study may be related to their higher densities, which tend to keep these particles submerged [ 44 ], increasing their contact with zebrafish. The environmental fate and degradation of each polymer is deeply influenced by its chemical composition [ 3 ]. Mechanical degradation and photodegradation of MPs result in the formation of macro radicals that may be formed in the presence of oxygen [ 45 ]. Plastics with carbon-carbon backbone, including PE, PP, PS, and PVC, are vulnerable to photo-initiated oxidative degradation and followed by chain scission reactions [ 46 ]. Among these, PS, with its aromatic rings, is especially susceptible to undergoing photo-oxidative breakdown, more than PE and PP, forming various oxygenated and unsaturated compounds, and is more prone to embrittlement [ 3 , 45 ]. PVC is highly sensitive to UV and thermal degradation, with dechlorination reactions producing reactive polyenes and fragmented particles [ 47 ]. On the other hand, PET’s ester bonds make it susceptible to both hydrolysis and photo-oxidation, broadening the pathways for surface reactivity [ 48 , 49 ]. Collectively, these properties dictate not just how microplastics persist and fragment, but also how they interact with and potentially impair aquatic organisms. Intrinsic polymer characteristics such as crystallinity, amorphous fraction, hydrophilicity, and density determine MPs persistence, fragmentation, and sinking behavior, as well as their interactions with aquatic organisms [ 43 , 48 ]. These properties influence settling dynamics and exposure potential, shaping the range of organisms and habitats that MPs contact. This may explain why, in our study, high-density polymers, PS, PET, and PVC, induced the most pronounced neuromast damage and behavioral impairments, reflecting the complex relationship between polymer chemistry, environmental fate, and biological impact in aquatic habitats. Similarly, the irregular surface of the MPs may increase mechanical abrasion of the cupula and hair bundles, physically damaging stereocilia and kinocilia, and reducing MET-dependent dye uptake [ 50 ]. MPs with rough surfaces and fragmentation resulting from mechanical degradation accelerate the leaching of additives, unreacted monomers, and oligomers into the environment by increasing the available surface area, potentially causing toxic effects [ 43 , 48 ]. Only a few studies have directly examined how plastic additives affect the zebrafish lateral line, and this remains an underexplored aspect of ototoxicity. Among them, TBBPA, a brominated flame retardant widely used in plastic formulations, has been reported to impair sensorimotor function and social behavior in larvae, consistent with neuromast dysfunction [ 51 ]. This sparse but growing evidence suggests that leaching additives, in addition to the polymer matrix itself, could contribute to the neuromast toxicity observed with household‑derived MPs, and highlights the limitations of the present study, and the need for future work explicitly targeting additive-lateral line interactions. In zebrafish, MPs impair neuromast function primarily by disrupting mechanotransduction, mitochondrial integrity, and redox balance. Our findings indicate that surface abrasion and sustained contact are key drivers of the observed mechanosensory deficits, whereas oxidative and leaching effects likely act as secondary contributors. Sublethal endpoints such as reduced escape responses and neuromast disruption may compromise ecological fitness even in the absence of overt mortality, while the reappearance of kinocilia in exposed larvae points to partial functional recovery supported by the robust regenerative capacity of zebrafish hair cells. These results support the zebrafish lateral line as a relevant new approach methodology (NAM) for sensory toxicology, offering high phenotypic resolution and molecular tractability to compare MPs with other ototoxic compounds that converge on conserved pathways, including impaired mechanotransduction, mitochondrial dysfunction, and oxidative stress. By integrating neuromast‑level phenotypes with gene‑expression and pathway‑level data, this model can feed into adverse outcome pathway frameworks and contribute to regulatory decision‑making on MP hazards, in line with NAM strategies that prioritize mechanistic, reductionist in vivo assays. In conclusion, MPs toxicity in developing zebrafish varied according to polymer type and fragment morphology, with PS, PET, and PVC producing the most prominent effects. The use of environmentally relevant fragments allowed us to assess developmental impacts, including reduced tactile responsiveness and compromised neuromast structure and function. Together, these findings demonstrate that acute exposure to MPs can disrupt mechanosensory systems during early life stages, underscoring the ecological relevance of neurosensory impairment as a sensitive endpoint. Overall, these results support the need for increased regulatory and scientific attention to behavioral and sensory endpoints in microplastic risk assessments. 4. Materials and methods 4.1 Microplastics synthesis Microplastic particles were generated from household products composed of polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC). The polymer type of each item was verified using resin identification codes. Items were thoroughly washed with distilled water, air-dried, and abraded using a metallic lime, modified from Villacorta et al. [ 52 ]. The resulting particles were collected and sieved through a 150 µm metallic mesh to isolate the desired size fraction, which was no larger than 150 µm. 4.2 FTIR-ATR spectroscopy The chemical identity of microplastics was verified using Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance (FTIR-ATR; Spectrum Two FT-IR Spectrometer, Perkin Elmer). Spectra were acquired from 4000 to 450 cm⁻¹ at a spectral resolution of 1 cm⁻¹ and 4 scans, modified from Villacorta et al. [ 52 ]. The ATR diamond was cleaned with isopropanol, and background spectra were collected between samples. For each polymer, 2 mg of microplastic was analyzed in triplicate, pressed at 80 N. Spectral analysis was performed using Spectragryph software (version 1.2.16.1) by averaging triplicate measurements and applying baseline correction with predetermined parameters. Polymer identification was based on characteristic absorption bands compared against published data [ 23 ] and the OpenSpecy database [ 24 ]. 4.3 Scanning electron microscopy (SEM) Microplastic morphology and particle size were evaluated using a LEICA Stereoscan 440 SEM operated at an accelerating voltage of 10 kV. Particles were mounted in SEM stubs prepared using carbon adhesive tape and dehydrated prior to imaging. SEM images were acquired at magnifications from 500x to 50,000x. For quantitative assessment, two representative images per sample were selected for particle counting and measurement (n PE = 16, PP = 28, PS = 41, PET = 20 and PVC = 32), which were scored using Fiji software [ 53 ]. 4.4 Zebrafish husbandry Wild-type and AB strain zebrafish ( Danio rerio ) were maintained at a fish husbandry in IBt, UNAM, Mexico, in a recirculating system at 28°C with a 14 h:10 h light: dark cycle [ 54 ]. Adults were fed daily with newly hatched Artemia nauplii and a commercial zebrafish diet (Skretting). For breeding, males and females were separated overnight and allowed to spawn the following morning. Eggs were collected approximately 40 minutes after fertilization, washed, and maintained in egg water (60 µg/mL Sea Salt, Instant Ocean) at 28°C until 4 hours post-fertilization (hpf) or sphere stage according to morphological criteria described by Kimmel et al. [ 12 ]. Zebrafish larvae were anesthetized with tricaine (3-aino benzoic acid ethyl ester) solution at a final concentration of 160mg/L in egg water. After completing all experiments, larvae were euthanized by immersion in ice-cold water. All experiments were performed in accordance with relevant guidelines and regulations approved by the Bioethical Committee (Instituto de Biotecnología, UNAM). Our study is reported in accordance with the ARRIVE guidelines. 4.5 Zebrafish embryo exposure and survival assessment Zebrafish embryo exposure was conducted with slight modifications to the Fish Embryo Acute Toxicity (FET) Test [ 55 ]. Briefly, at the sphere stage, embryos were transferred to 48-well plates (10 embryos per well) containing 300 µL of a 1 mg/mL microplastic suspension in egg water, with one polymer per treatment group. Plates were incubated at 28°C in a humidified chamber. Embryo survival, morphology (compared to that reported by Kimmel et al. [ 12 ]), and hatching were monitored daily using a ZEISS Stemi 508 stereomicroscope. Each exposure experiment was carried out in triplicate. 4.6 Touch-evoked response Touch-evoked escape responses were assessed at 72, 96, and 120 hpf, following a modified version of Sztal et al.[ 56 ]. Larvae were individually placed in the center of a 10 mm culture dish containing egg water at 28°C, acclimated for 1 minute, and gently stimulated on the trunk with a blunt tip glass probe (up to 10 stimuli per cycle, with 30 seconds rest between cycles). If a larva failed to respond after five cycles, the assay was concluded. A positive escape response was defined as any swimming movement following tactile stimulation. Approximately 30 larvae were analyzed per treatment. Responses were video recorded using a Zeiss Stemi 508 stereomicroscope. Videos were analyzed with manual tracking using Fiji software for the number of stimuli required, swim distance, and active response time. 4.7 SEM sample preparation: zebrafish larvae Larvae at 96 and 120 hpf were fixed in 2.5% glutaraldehyde for 2 hours at 4°C, followed by three 15-minute washes in cold PBS. Post-fixation was performed in osmium tetroxide for 1 hour, followed by three further PBS washes (15 minutes each). Samples were dehydrated on ice using a graded ethanol series (40%–100%, 10% increments, 15 minutes per step). SEM stubs were cleaned and prepared with carbon adhesive tape during dehydration. Dehydrated larvae were mounted on stubs and gold-coated using an ion sputter coater (10 minutes). Imaging was conducted on a LEICA Stereoscan 440 SEM at 10 kV with preparations stored under vacuum until observation. Images were acquired at 100x, 5000x, 10000x, and 25000x. 4.8 Fluorescent staining and confocal imaging Neuromasts were stained as described by Owens et al. [ 57 ]. Staining was carried out at 72, 96, and 120 hpf with 0.05% DASPEI (Invitrogen, CA, US) in egg water for 30 minutes, followed by a 1-minute wash and a 5-minute egg water wash. Cell death was detected with 10 µg/mL acridine orange (AO) (Invitrogen, CA, US) for 30 minutes, followed by three 10-minute washes in egg water, modified from Mendieta Serrano et al. [ 58 ]. Bodipy TR (5 µg/mL, Invitrogen) was used for additional staining after AO. All incubations were performed in the dark. Larvae were anesthetized as described previously and embedded in 1.5% low-melting-point agarose and imaged with an Olympus IX81 inverted confocal microscope using 20x and 60x objectives. DASPEI and AO were excited at 488 nm, and Bodipy TR at 543 nm. 4.9 Statistical analysis Normality was assessed using the Shapiro-Wilk test. Comparisons between control and each treatment groups were performed using unpaired t-tests were for normally distributed data and Welch’s t-tests for non-normal data. Statistical significance was considered at p < 0.05. Analyses were conducted in GraphPad Prism (version 10) with 8–10 embryos included in each replicate each experiment was conducted in triplicate. Declarations Competing interests The author(s) declare no competing interests Funding The research was funded by the PAPIIT DGAPA UNAM program IN208122 and IN222325 Author Contribution ACM: Formal análisis, Investigation, Methodology, Writing – original draft, Writing – review & editing. NMM: Formal análisis, Methodology Writing – review & editing. LPL: Writing – review & editing..TMUR: Methodology, Writing – review & editing. IHO: Writing – review & editing. HL:Formal análisis, Supervision Writing – review & editing. ADVR: Conceptualization, Formal análisis, Supervision, Writing – review & editing, DSP: ConceptualizationFormal análisis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing Acknowledgement We thank Enrique Salas and Arlen Ramirez Corona (IBt, UNAM) for their technical support in the acquisition of confocal images. 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1","display":"","copyAsset":false,"role":"figure","size":2020634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynthesized microplastic fragments correspond to the expected polymer.\u003c/strong\u003e Representative FTIR-ATR spectra show the diagnostic absorbance peaks for PE, PP, PS, PET, and PVC, allowing chemical identification of each polymer. Absorbance peaks indicative of each polymer is highlighted. Statistical analysis: Mann-Whitney U test; **p \u0026lt; 0.01, **p \u0026lt; 0.0001. Abbreviations: FTIR, Fourier Transform Infrared Spectroscopy; ATR, Attenuated Total Reflectance; PE, polyethylene; PP, polypropylene; PS, polystyrene; PET, polyethylene terephthalate; PVC, polyvinyl chloride.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/91852b2476d738744a1a4ed0.png"},{"id":100755452,"identity":"00b4995f-d928-4d3a-8806-c1a6e6e69b8f","added_by":"auto","created_at":"2026-01-21 06:20:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4976318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroplastics show heterogeneous morphology and size.\u003c/strong\u003e SEM micrographs (A–E) display the surface features of PE, PP, PS, PET, and PVC microplastics, showing irregular and rough-edged fragment shapes. Bar 100 µm. (F) Particle length distribution for each polymer, determined from SEM image analysis. Statistical analysis: Mann-Whitney U test; **p \u0026lt; 0.01, **p \u0026lt; 0.0001. Abbreviations: SEM, Scanning Electron Microscopy; PE, polyethylene; PP, polypropylene; PS, polystyrene; PET, polyethylene terephthalate; PVC, polyvinyl chloride.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/f8c5e167dc5e82951a548d14.png"},{"id":100755474,"identity":"d7002bdf-8276-4260-8fc7-65f23497dca4","added_by":"auto","created_at":"2026-01-21 06:20:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":456532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroplastic exposure reduces touch-evoked escape responses in developing zebrafish larvae.\u003c/strong\u003e (A) Bar graphs show the percentage of larvae exhibiting a positive response to tactile stimulation for the control group and each microplastic treatment (PE, PP, PS, PET, PVC) at 72, 96, and 120 hpf. Bars indicate mean ± SEM for each group. (B) violin plots display the number of stimuli required to elicit a response in individual larvae at each time point. Statistical analysis was performed using the Welch T test; *p \u0026lt; 0.05, **p \u0026lt; 0.01. Abbreviations: PE, polyethylene; PP, polypropylene; PS, polystyrene; PET, polyethylene terephthalate; PVC, polyvinyl chloride; hpf, hours post-fertilization; SEM, standard error of the mean.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/1a1b13feb0b8bd21bc59c95e.png"},{"id":100755470,"identity":"5373793f-2802-478e-aa0d-7bfcbf507ef4","added_by":"auto","created_at":"2026-01-21 06:20:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20851990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZebrafish neuromasts showed mechanical disruption or kinocilia fusionof the hair cells when exposed to diverse microplastics.\u003c/strong\u003e SEM imaging reveals direct microplastic contact and associated damage on the exterior surface of. SEM micrographs taken at 96 and 120 hpf display neuromast morphology on the lateral surface of larvae from control and each microplastic exposure group (PE, PP, PS, PET, PVC). For each condition, a magnified inset to the right provides a close-up view of the interaction between microplastic fragments and neuromast structures. Bar 5 mm. Asterisks mark the locations of microplastics in each image. Head arrows show the fusion of the kinocilia Abbreviations: SEM, Scanning Electron Microscopy; PE, polyethylene; PP, polypropylene; PS, polystyrene; PET, polyethylene terephthalate; PVC, polyvinyl chloride; hpf, hours post-fertilization.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/451d262f293f96efefec2e61.png"},{"id":100755418,"identity":"a0642689-3edc-46bd-924f-04f933a42ec2","added_by":"auto","created_at":"2026-01-21 06:20:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8098243,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroplastic exposure reduces neuromast number in the posterior lateral line of zebrafish larvae.\u003c/strong\u003e (A) Representative DASPEI-stained images of whole-mount zebrafish larvae at 96 and 120 hpf show neuromast labeling in the control group and single larva exposed to MP, highlighting differences in neuromast distribution (asterisk) Bar 0.5 mm. (B) Box and (C) whisker plots indicate the number of posterior lateral line (PLL) neuromasts for each group (control and all MP treatments) at 96 and 120 hpf. Statistical analysis was performed using the Welch t-test comparing control to each MP treatment; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Abbreviations: DASPEI, 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide; MP, microplastics; PE, polyethylene; PP, polypropylene; PS, polystyrene; PET, polyethylene terephthalate; PVC, polyvinyl chloride; hpf, hours post-fertilization; PLL, posterior lateral line.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/67b42b3ea04c0e12288d5ff3.png"},{"id":100755438,"identity":"da8164a7-fcab-42c0-8333-d7b87a2f1b3d","added_by":"auto","created_at":"2026-01-21 06:20:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1880421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondrial network disrupted in neuromast hair cells following microplastic exposure\u003c/strong\u003e. DASPEI-stained confocal images of zebrafish neuromasts at 96 and 120 hpf from control and each microplastic treatment group (PE, PP, PS, PET, PVC) display hair cell mitochondrial architecture. Each panel shows a representative neuromast; in the corner of each panel displays the numbers of neuromasts that showed that morphology of different treatments from the total of neuromasts observed. Bar 10 mm. Asterisk show the mitochondrial network disrupted. Abbreviations: DASPEI, 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide; PE, polyethylene; PP, polypropylene; PS, polystyrene; PET, polyethylene terephthalate; PVC, polyvinyl chloride; hpf, hours post-fertilization.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/e5c61b20304f0f55ee05f1e3.png"},{"id":100755436,"identity":"5638b241-3411-420d-9a0c-01b82a2b65f9","added_by":"auto","created_at":"2026-01-21 06:20:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":11902383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe general structure of the neuromast cells remains unaffected after MP exposure.\u003c/strong\u003e Confocal micrographs of neuromasts at 96 and 120 hpf show both control and microplastic-treated larvae. Each panel includes images marking apoptotic cells and cell membranes, as well as merged images for each treatment with red and green respectively. While the cell membrane stain highlights neuromast morphology and structural changes, the apoptotic cell signal appears similar across groups, indicating no detectable increase in cell death due to microplastic exposure. Bar 10 mm Abbreviations: BODIPY-TR, boron-dipyrromethene tetramethylrhodamine; hpf, hours post-fertilization.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/ba3d33ce0a3d3986da385fb1.png"},{"id":103049084,"identity":"0076b5ae-6f41-49b3-b66f-f51b2cf4e056","added_by":"auto","created_at":"2026-02-20 07:29:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45984944,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/0006599a-42ff-4aca-aa27-e8523439003f.pdf"},{"id":100755423,"identity":"75db114f-8812-4e2d-9715-2bad85b88875","added_by":"auto","created_at":"2026-01-21 06:20:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":349507,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialCazaresMoralesA2025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8398009/v1/fad0dbd80c98f1df4f643026.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Behavioural impact of microplastics on zebrafish development","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlastic production has increased exponentially since its widespread use in the 1950s, with more than half of all plastic ever produced being generated between 2000 and 2020 alone [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As the use and disposal of plastic escalates, and because of its slow degradation rate, the persistent accumulation of plastic debris is observed in both aquatic and terrestrial environments [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Moreover, particularly in marine ecosystems, larger plastic items are subject to photooxidation and mechanical degradation, fragmenting into microplastics [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroplastics (MPs) are particles smaller than 5 mm and are highly heterogeneous, varying in polymer composition, size, colour, and shape [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These features influence their biological activity and environmental fate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although MPs are generally considered non-lethal at environmental concentrations, growing evidence links MP exposure to a broad spectrum of adverse biological effects, including neurodevelopmental and immune dysfunction, endocrine disruption, gastrointestinal disturbances, metabolic and cardiovascular impairments, oxidative stress, and microbiota dysbiosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Notably, MPs have recently been detected in human placental tissue, meconium, and breast milk, raising concerns about the potential adverse effects of MP exposure during early development [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMost experimental studies investigating microplastic toxicity have relied primarily on pristine, laboratory-generated particles\u0026mdash;typically uniform spheres\u0026mdash;composed of a single polymer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This approach contrasts sharply with environmental reality, where microplastics detected in air, water, and food are predominantly heterogeneous fibers and fragments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The exclusive use of pristine spheres and limited polymer types in toxicological studies can underestimate or misrepresent the true ecological risks posed by microplastics. It has been suggested that toxicity increases with decreased particle size and is further amplified by specific shapes. Fibers are often more hazardous than fragments, which in turn are more toxic than spheres [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Fibers and fragments, often weathered and chemically altered, exhibit greater toxicity and are more environmentally relevant, reflecting the diversity and complexity of real-world exposures [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, toxicological studies have disproportionately focused on a few MPs, overlooking the diversity and weathering found in environmental samples [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This approach can have profound consequences as physicochemical properties, such as polymer type, shape, size, colour, surface chemistry, molecular structure, density, and environmental aging, all influence toxicological outcomes, affecting MPs' bioactivity, sorption capacities, and interactions with organisms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The assessment of a broader range of microplastic shapes and chemical compositions is therefore essential to provide meaningful insights into the health of aquatic organisms and to improve environmental risk assessment studies.\u003c/p\u003e \u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) embryos and larvae have emerged as a powerful tool in MP research, due to their rapid external development, optical transparency, and high genetic and physiological homology to humans [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Zebrafish produce hundreds of offspring per clutch, enabling large sample sizes and robust statistical analyses [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Their direct exposure to waterborne MPs mirrors realistic environmental conditions and enables sensitive detection of effects across developmental, behavioural, and molecular endpoints [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrent scientific reports reveal a spectrum of MP-related effects in zebrafish, from early hatching or reduced survival [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], developmental changes in morphometric parameters [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and cardiotoxicity, to downregulation of nervous system and metabolic gene pathways [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Behavioural studies describe seizure-like activity, loss of swimming competence, and altered neurochemical profiles [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. MPs have also been implicated in disrupting the microbiome [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], compromising visual system integrity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and causing genotoxicity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Importantly, some studies report adverse outcomes even at the lowest environmentally relevant concentrations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], while others note little influence on growth or hatching, detecting only changes in gene expression or subtle physiological alterations [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Nevertheless, most studies performed in zebrafish have mainly used polystyrene (PS) spheres [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, we systematically produced and characterized a MPs representing those found in environmental samples, such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC), polymers of everyday household items [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Zebrafish embryos were exposed to these MPs from 4 to 120 hours post-fertilization, with assessments of survival, hatching, touch-evoked escape behaviour, and motility. While general swimming capacity was unaffected, exposure impaired the touch-evoked response, prompting focused investigation on neuromast integrity and cell death. The neuromasts of zebrafish are specialized sensory organs that form part of the lateral line system, enabling the detection of water movements and vibrations in the surrounding environment. Neuromasts develop during embryogenesis from migrating primordia that deposit clusters of cells at stereotypical positions along the head and body surface [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Each neuromast consists of mechanosensory hair cells (analogous to those in the mammalian inner ear) interspersed with supporting and mantle cells, all covered by a gelatinous cupula that transmits mechanical stimuli to the hair cells underneath [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The precise arrangement and continual regeneration of hair cells within each neuromast provides zebrafish with robust and adaptable flow-sensing capabilities, underlying essential behaviours such as rheotaxis, prey detection, predator avoidance, and schooling [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The accessibility and regenerative capacity of lateral line neuromasts make them an invaluable model for studying hair cell biology, sensory organ development, and environmental toxicology.\u003c/p\u003e \u003cp\u003eBy mapping hazardous outcomes across developmental and sensory endpoints and comparing polymers, our findings contribute to a more detailed understanding of MP toxicology, essential for assessing ecological risks and guiding policy amid the growing plastic pollution crisis. Identifying and understanding the impacts of MP on early-life development is vital, not only for aquatic organisms but also for informing concerns about potential risks during pregnancy and for future generations. The complex interplay of particle characteristics, environmental modification, and biological response demands that toxicological studies reflect realistic exposures by including diverse, environmentally relevant MP polymers and morphologies.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Characterization of synthesized microplastics\u003c/h2\u003e \u003cp\u003eThe identity of the five synthesized microplastic samples, polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC), was confirmed by FTIR-ATR spectroscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, supplementary Table\u0026nbsp;1). Characteristic absorption bands for each polymer were compared with reference values from [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and spectral matches in the OpenSpecy database [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The PE, PP, PS, and PET samples demonstrated absorption bands that either directly matched or fell within 3 wavenumbers of the literature values (supplementary Table\u0026nbsp;1). Spectral correlation analysis using OpenSpecy yielded high match scores for PE, PP, PS, and PET to published spectra (r\u0026thinsp;=\u0026thinsp;0.99, Primpke et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]). For PVC, differences in the main bands were observed at 1242, 1094, and 611 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, although the database still indicated a positive match (r\u0026thinsp;=\u0026thinsp;0.84) consistent with De Frond et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While all polymers could be matched, the PVC sample showed the lowest spectral agreement, likely due to the presence of additives or plasticizers, as differences in the 1500\u0026ndash;600 cm\u003csup\u003e1\u003c/sup\u003e region are often observed when such unbound compounds are present [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccurate determination of particle size is important as particle dimensions strongly influence environmental fate, biological interactions, and potential toxicity of MPs. Size affects sedimentation, bioavailability, and the likelihood of tissue contact or cellular uptake, making robust size characterization essential for interpreting toxicological outcomes and comparing results across studies. SEM imaging further revealed the surface morphology and size distribution of the synthesized microplastics (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The particles exhibited irregular, rough-edged fragments with occasional fibrous structures, consistent with mechanical abrasion used as the preparation method. Median particle length differed by polymer, with PS fragments being the largest (172.87 \u0026micro;m, Q1: 140.22, Q3: 235.46), followed by PVC (171.14 \u0026micro;m, Q1: 123.65, Q3: 245.88), PE (161.72 \u0026micro;m, Q1: 142.25, Q3: 221.93), PP (108.97 \u0026micro;m, Q1: 82.83, Q3: 134.56), and PET (101.62 \u0026micro;m, Q1: 82.83, Q3: 134.56). Pairwise comparisons revealed that PE fragments were significantly longer than PET (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not different from PP, PS, or PVC. PP fragments were shorter than PS (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and PVC (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while no significant differences were detected between PP and PET. PS fragments were longer than PET (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and PP, but did not differ from PVC. PET fragments were shorter than PVC (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These findings indicate clear, polymer-dependent heterogeneity in the size distributions of environmentally relevant MP fragments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, the combined FTIR-ATR and SEM analyses confirm the successful synthesis and accurate identification of the five microplastic types. The chemical and morphological characteristics observed are consistent with the literature. We were able to produce irregular MPs similar to those found in environmental samples, supporting their use in subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microplastic effect in Zebrafish development\u003c/h2\u003e \u003cp\u003eZebrafish larvae were exposed to the different types of microplastics. No significant differences were observed in survival, hatching rates, or gross morphology between exposed and control groups (Supplementary Fig.\u0026nbsp;1). However, a touch-evoked response test showed that microplastics caused a reduction in larvae\u0026rsquo;s response. We conducted touch-evoked response assays at 72, 96, and 120 hpf (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Larvae exposed to microplastics exhibited diminished sensitivity, requiring more tactile stimuli to elicit an escape response. Approximately 80% of control larvae responded to the first stimulus, and the rest responded to a second stimulus at all the times tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). At 72hpf, PE-treated larvae reacted similarly to the controls, while the rest of the MP treatments showed that only about half of the larvae responded at the first touch (PP 60.83%; PS 53.85%; PET 59.35% and PVC 49.1%; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), requiring four to eight stimuli to respond (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At 96 hpf, all the MPs showed that at least 50% of the larvae are affected in the touch-evoked response (PE 56.48%; PP 52.06%; PS 52.22%; PET 54.23% and PVC 38.33%). At 96 hpf, we also observed that the larvae required a higher number of stimuli to react and that in some cases, larvae did not react after ten stimuli when the test ended. By 120 hpf, 83.33% of controls reacted to the first stimulus, and PE and PP groups showed no significant difference from controls; nevertheless, an increased number of stimuli was still observed compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), while response rates for PS, PET, and PVC remained below 60% (PE 76.25%; PP 77.96%; PS 50%; PET 58.51% and PVC 58.33%) significantly different from controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImportantly, once the escape response was initiated, swimming performance and activity duration were comparable to those of controls (Supplementary Fig.\u0026nbsp;2). These results indicate that, under experimental conditions, microplastic exposure affected the larvae\u0026rsquo;s sensitivity to tactile stimuli but did not impair neuromotor or motor function. Because altered tactile responsiveness can arise from disruptions in lateral line mechanoreceptors, we next assessed the structural integrity of neuromasts.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Neuromast characterization\u003c/h2\u003e \u003cp\u003eTo assess the impact of MPs on mechanosensory integrity, the structure of lateral line neuromasts was examined following exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). SEM analysis at 96 and 120 hpf revealed that neuromasts in untreated controls possessed large, distinct kinocilia. In larvae exposed to PE and PP, kinocilia were absent at 96 hpf, though short kinocilia appeared in the PE and PP groups at 120 hpf. In contrast, larvae treated with PS, PET, or PVC displayed predominantly short kinocilia at 96 hpf, with evidence of possibly kinocilia fusion by 120 hpf. Microplastic particles were frequently observed adhering near or directly to neuromasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further characterize the lateral line neuromasts, we used DASPEI staining. The vital dye DASPEI labels hair cell mitochondria in the neuromast and is used to assess their integrity. Our studies demonstrated a significant reduction in neuromast number in the lateral line at 96 hpf for MP-treated groups: while control larvae averaged 8 to 9 neuromasts, exposed groups had only 6 to 7, and counts as low as 4 were observed in some PS or PVC-exposed larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). By 120 hpf, the PE, PP, and PVC groups showed partial recovery in neuromast number, whereas the PS and PET groups remained lower than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt higher magnification (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), control neuromasts showed hair cells arranged in a convergent, flower-like configuration, with robust and elongated (mitochondria-rich) DASPEI-positive hair cells. At 96 hpf, larvae exposed to MPs displayed weaker fluorescence intensity in neuromast hair cells, indicative of reduced hair cell density or integrity. By 120 hpf, increased DASPEI staining was noted in PE- and PVC-treated larvae compared to 96 hpf, although mitochondria appeared shorter than the typical elongated architecture observed under normal conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Fluorescence was generally weaker with PS and PET exposure than in controls. Incomplete or patchy staining was particularly pronounced at 96 hpf for all MP groups, and only partial recovery of hair cell structure was observed by 120 hpf of PE and PVC, with the mitochondrial network less distinct than in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the integrity of the neuromast hair cells, we conducted dual staining with acridine orange to detect cell death and Bodipy to visualize the membranes of the neuromast cells at 96 and 120 hpf. The staining revealed no significant differences in hair cell death within neuromasts between control and MP-exposed larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Further staining with Bodipy enabled visualization of both hair cells and supporting cells within the neuromast, with no evident architectural alterations between treated and control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, although acridine orange staining revealed no significant differences in cell death between control and microplastic-exposed groups, collectively, exposure to microplastics during early zebrafish development disrupts the function of lateral line neuromasts. Marked alterations were consistently observed in neuromast cilia morphology and mitochondrial network that alter the function in the exposed larvae, consistent with the lack of touch-evoke response in the treated zebrafish larvae. Changes in kinocilia morphology and reduced numbers of DASPEI-positive hair cells suggest impaired mechanosensory function. The persistence of these defects, even with some recovery at later stages, highlights the sensitivity of neuromasts to microplastic-induced damage and underscores the risk to sensory development and function in aquatic organisms exposed to environmentally relevant levels of microplastics.\u003c/p\u003e \u003cp\u003eTaken together, these findings indicate that acute exposure to environmentally relevant, irregular microplastic fragments causes significant sublethal effects in developing zebrafish. Larvae showed reduced touch-evoked escape responses and clear disruption of lateral line neuromasts, including mechanical damage, kinocilia fusion, and decreased mitochondrial activity. All tested polymers induced damaged neuromast integrity, with PS, PET, and PVC causing the strongest effects. These results suggest that material properties and fragment shape are key factors in neurosensory toxicity.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eMicroplastic pollution in aquatic environments is associated with a broad spectrum of biological effects in zebrafish, including neurodevelopmental toxicity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], gastrointestinal disturbance [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], microbiota dysbiosis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], cardiac dysfunctions [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and metabolic imbalances [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. While these adverse outcomes are well documented, the mechanisms underlying sublethal neurotoxicity and physical damage, especially at early developmental stages, remain poorly understood.\u003c/p\u003e \u003cp\u003eIn contrast to most studies that use pristine, single polymers, our work used irregular MPs generated from common household materials, capturing the heterogeneity of polymer types and surface features encountered in real-world conditions. Although the exposure concentration (1 mg/mL) may seem high compared to most levels reported in environmental matrices, it was selected to represent a conservative worst-case scenario and to ensure detection of polymer-specific effects that could otherwise be absent at lower doses. Nevertheless, some reports indicate that MPs can reach or exceed the concentration applied here in highly impacted contexts; MP concentrations of 1.8 mg/mL have been reported in wastewater from a plastic recycling facility [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], demonstrating the potential for extremely elevated local contamination levels; MP surface loadings of 3.63 mg/cm\u0026sup2; have been reported along the Canary Islands coastline [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], indicating substantial accumulation of MPs in coastal depositional zones.; and MP concentrations in human liver and brain have been reported to increase significantly between 2016 and 2024, with brain levels in 2024 reaching 4.917 mg/g and higher burdens observed in individuals with dementia than in healthy controls [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Taken together, these data suggest that although our exposure concentration could exceed typical environmental levels, it falls within the range observed in heavily contaminated systems and in human tissues, supporting the toxicological and hazard-oriented relevance of the dose used in our experiments.\u003c/p\u003e \u003cp\u003eThe zebrafish lateral line is an increasingly used model for investigating ototoxicity and is a suitable alternative to other vertebrate models, as it reproduces ototoxic responses similar to those observed in humans exposed to various compounds [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Neuromasts are the lateral line sensory organs that detect water movement and enable behaviors like predator avoidance and schooling [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Each neuromast contains mechanosensory hair cells whose stereocilia and a kinocilium deflect with water flow, opening the mechanoelectrical transduction (MET) channels, depolarizing the cell, and triggering calcium-dependent neurotransmitter release onto afferent neurons [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Because hair cells have high energetic demands, they are particularly vulnerable to mitochondrial disruption. In neuromast hair cells, mitochondria occupy a substantial portion of the cell and form an extensive, interconnected network [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Proper mechanotransduction is essential for the development of this specialized mitochondrial architecture, while ongoing synaptic transmission promotes the growth and specific localization of large mitochondria. Disruption of mechanotransduction or synaptic activity can alter mitochondrial structure and compromise hair cell function [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe impaired touch-evoked escape responses observed in our study suggest that household-derived MPs compromise mechanotransduction in zebrafish larvae. The reduced DASPEI labeling may reflect either inhibition of MET channel function or degeneration of hair bundle cilia. Exposure to MPs resulted in striking morphological alterations kinocilia appeared fused or tangled and the number and intensity of DASPEI-positive hair cells were reduced, particularly after PS and PET treatment. Our results are consistent with previous findings that large MP fragments and high doses cause agglomeration of hair cell bundles [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These results are also consistent with impaired MET channel function, as fused hair bundles likely hinder channel opening, thereby restricting dye entry and decreasing fluorescence [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] causing a decline at the cation influx necessary to induce a signal.\u003c/p\u003e \u003cp\u003eNeuromasts contain a mosaic of both mature and young hair cells [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and MP exposure may disrupt this homeostatic renewal, compounding sensory deficits over developmental time. In MP‑treated neuromasts, we observed fragmented, abnormally short mitochondria, whereas control larvae displayed the typical architecture with numerous large basal mitochondria supporting synaptic function and smaller apical mitochondria; damage to this network is associated with reduced membrane potential, decreased ATP production, and increased susceptibility to apoptosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Notably, in some neuromasts, the cilia that were absent at earlier time points appeared again, consistent with the ongoing renewal and regeneration of hair cells in the lateral line.\u003c/p\u003e \u003cp\u003eOtotoxic compounds provide a useful framework for interpreting the neuromast damage observed in MP‑exposed larvae, as many of the structural and functional alterations resemble classic hair‑cell ototoxicity patterns described in the zebrafish lateral line model. Metals such as copper, manganese, and cobalt rapidly impair mechanotransduction and neuromast function, either by disrupting stereocilia organization and blocking MET channel, or by decreasing afferent neuron sensitivity, with high copper doses even preventing regeneration [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Similarly, antibiotics (neomycin) and antitumoral drugs (cisplatin) induce hair‑cell death through combined disruption of Ca\u0026sup2;⁺ homeostasis in the endoplasmic reticulum\u0026ndash;mitochondria dynamic, mitochondrial overload, and excessive ROS generation, ultimately leading to loss of membrane potential, activation of apoptotic cascades [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Within this ototoxic context, the reduced DASPEI staining, impaired touch‑evoked responses, and hair‑bundle abnormalities caused by household‑derived MPs align with a broader paradigm in which diverse xenobiotics converge on a common target, mechanotransduction channels, mitochondrial integrity, and oxidative balance to compromise neuromast function and sensorimotor performance.\u003c/p\u003e \u003cp\u003eOxidative stress is among the most frequently reported adverse effects associated with exposure to MPs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. During MP breakdown, free radicals are generated within the polymer chains and can react with oxygen to form additional reactive species. The chemical structure and composition of each polymer play a crucial role in determining its susceptibility to free radical formation during weathering and degradation in the marine environment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mitochondria-rich hair cells are particularly vulnerable to oxidative stress and high metabolic demands [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Thus, the observed loss of mechanosensory function and morphologic integrity in lateral line neuromasts is likely mediated by a combination of mechanical abrasion, impaired ion transport, mitochondrial dysfunction, and oxidative damage, culminating in impaired sensorimotor and behavioral capacity in exposed zebrafish larvae. Hydrogen peroxide exposure has been reported to damage neuromasts by inducing kinociliary abnormalities, promoting hair cell detachment from the neuromast rosette, and ultimately leading to hair cell death [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Such oxidative stress disrupts the structural and functional integrity of hair cells, diminishing their ability to detect mechanical stimuli and compromising overall sensory performance.\u003c/p\u003e \u003cp\u003eAlthough oxidative stress was not directly measured in this study, its contribution to the observed effects cannot be ruled out. Other studies in marine, freshwater, and terrestrial organisms, including fish, crustaceans, mollusks, and rotifers, reported that PE, PP, PS, PET, and PVC can induce both oxidative stress and neurotoxicity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The evidence indicates that microplastics primarily induce neuromast damage through mechanical abrasion of the epithelial surface, as the relatively large fragment size used prevents cellular internalization. The results are best explained by physical contact and surface injury rather than internalized chemical mechanisms. Notably, PS, PET, and PVC were associated with the most pronounced neuromast and behavioral impairments. This pattern does not strictly follow the conventional idea that smaller MPs are always more toxic; PS in our study represented the largest particle size yet induced severe effects, while PVC was among the smallest and caused marked toxicity. Fragment shape did not vary significantly across polymer types, pointing to physicochemical differences as key explanatory factors. While additional factors such as density, surface hardness, and hydrophobicity are established in the literature as important determinants of microplastic toxicity, their impacts are inferred here based on the known properties of each polymer and are not directly measured. Therefore, our findings highlight that even among irregular fragments of similar shape, polymer-specific composition, and resulting differences in particle\u0026ndash;tissue interactions play a central role in the observed patterns of neuromast damage and behavioral impairment.\u003c/p\u003e \u003cp\u003eThe enhanced impact of PS, PET, and PVC in our study may be related to their higher densities, which tend to keep these particles submerged [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], increasing their contact with zebrafish. The environmental fate and degradation of each polymer is deeply influenced by its chemical composition [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mechanical degradation and photodegradation of MPs result in the formation of macro radicals that may be formed in the presence of oxygen [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Plastics with carbon-carbon backbone, including PE, PP, PS, and PVC, are vulnerable to photo-initiated oxidative degradation and followed by chain scission reactions [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Among these, PS, with its aromatic rings, is especially susceptible to undergoing photo-oxidative breakdown, more than PE and PP, forming various oxygenated and unsaturated compounds, and is more prone to embrittlement [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. PVC is highly sensitive to UV and thermal degradation, with dechlorination reactions producing reactive polyenes and fragmented particles [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. On the other hand, PET\u0026rsquo;s ester bonds make it susceptible to both hydrolysis and photo-oxidation, broadening the pathways for surface reactivity [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Collectively, these properties dictate not just how microplastics persist and fragment, but also how they interact with and potentially impair aquatic organisms.\u003c/p\u003e \u003cp\u003eIntrinsic polymer characteristics such as crystallinity, amorphous fraction, hydrophilicity, and density determine MPs persistence, fragmentation, and sinking behavior, as well as their interactions with aquatic organisms [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These properties influence settling dynamics and exposure potential, shaping the range of organisms and habitats that MPs contact. This may explain why, in our study, high-density polymers, PS, PET, and PVC, induced the most pronounced neuromast damage and behavioral impairments, reflecting the complex relationship between polymer chemistry, environmental fate, and biological impact in aquatic habitats. Similarly, the irregular surface of the MPs may increase mechanical abrasion of the cupula and hair bundles, physically damaging stereocilia and kinocilia, and reducing MET-dependent dye uptake [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. MPs with rough surfaces and fragmentation resulting from mechanical degradation accelerate the leaching of additives, unreacted monomers, and oligomers into the environment by increasing the available surface area, potentially causing toxic effects [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOnly a few studies have directly examined how plastic additives affect the zebrafish lateral line, and this remains an underexplored aspect of ototoxicity. Among them, TBBPA, a brominated flame retardant widely used in plastic formulations, has been reported to impair sensorimotor function and social behavior in larvae, consistent with neuromast dysfunction [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. This sparse but growing evidence suggests that leaching additives, in addition to the polymer matrix itself, could contribute to the neuromast toxicity observed with household‑derived MPs, and highlights the limitations of the present study, and the need for future work explicitly targeting additive-lateral line interactions.\u003c/p\u003e \u003cp\u003eIn zebrafish, MPs impair neuromast function primarily by disrupting mechanotransduction, mitochondrial integrity, and redox balance. Our findings indicate that surface abrasion and sustained contact are key drivers of the observed mechanosensory deficits, whereas oxidative and leaching effects likely act as secondary contributors. Sublethal endpoints such as reduced escape responses and neuromast disruption may compromise ecological fitness even in the absence of overt mortality, while the reappearance of kinocilia in exposed larvae points to partial functional recovery supported by the robust regenerative capacity of zebrafish hair cells.\u003c/p\u003e \u003cp\u003eThese results support the zebrafish lateral line as a relevant new approach methodology (NAM) for sensory toxicology, offering high phenotypic resolution and molecular tractability to compare MPs with other ototoxic compounds that converge on conserved pathways, including impaired mechanotransduction, mitochondrial dysfunction, and oxidative stress. By integrating neuromast‑level phenotypes with gene‑expression and pathway‑level data, this model can feed into adverse outcome pathway frameworks and contribute to regulatory decision‑making on MP hazards, in line with NAM strategies that prioritize mechanistic, reductionist \u003cem\u003ein vivo\u003c/em\u003e assays.\u003c/p\u003e \u003cp\u003eIn conclusion, MPs toxicity in developing zebrafish varied according to polymer type and fragment morphology, with PS, PET, and PVC producing the most prominent effects. The use of environmentally relevant fragments allowed us to assess developmental impacts, including reduced tactile responsiveness and compromised neuromast structure and function. Together, these findings demonstrate that acute exposure to MPs can disrupt mechanosensory systems during early life stages, underscoring the ecological relevance of neurosensory impairment as a sensitive endpoint. Overall, these results support the need for increased regulatory and scientific attention to behavioral and sensory endpoints in microplastic risk assessments.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Microplastics synthesis\u003c/h2\u003e \u003cp\u003eMicroplastic particles were generated from household products composed of polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET), and polyvinyl chloride (PVC). The polymer type of each item was verified using resin identification codes. Items were thoroughly washed with distilled water, air-dried, and abraded using a metallic lime, modified from Villacorta et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The resulting particles were collected and sieved through a 150 \u0026micro;m metallic mesh to isolate the desired size fraction, which was no larger than 150 \u0026micro;m.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.2 FTIR-ATR spectroscopy\u003c/h2\u003e \u003cp\u003eThe chemical identity of microplastics was verified using Fourier Transform Infrared Spectroscopy with Attenuated Total Reflectance (FTIR-ATR; Spectrum Two FT-IR Spectrometer, Perkin Elmer). Spectra were acquired from 4000 to 450 cm⁻\u0026sup1; at a spectral resolution of 1 cm⁻\u0026sup1; and 4 scans, modified from Villacorta et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The ATR diamond was cleaned with isopropanol, and background spectra were collected between samples. For each polymer, 2 mg of microplastic was analyzed in triplicate, pressed at 80 N. Spectral analysis was performed using Spectragryph software (version 1.2.16.1) by averaging triplicate measurements and applying baseline correction with predetermined parameters. Polymer identification was based on characteristic absorption bands compared against published data [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and the OpenSpecy database [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Scanning electron microscopy (SEM)\u003c/h2\u003e \u003cp\u003eMicroplastic morphology and particle size were evaluated using a LEICA Stereoscan 440 SEM operated at an accelerating voltage of 10 kV. Particles were mounted in SEM stubs prepared using carbon adhesive tape and dehydrated prior to imaging. SEM images were acquired at magnifications from 500x to 50,000x. For quantitative assessment, two representative images per sample were selected for particle counting and measurement (n PE\u0026thinsp;=\u0026thinsp;16, PP\u0026thinsp;=\u0026thinsp;28, PS\u0026thinsp;=\u0026thinsp;41, PET\u0026thinsp;=\u0026thinsp;20 and PVC\u0026thinsp;=\u0026thinsp;32), which were scored using Fiji software [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Zebrafish husbandry\u003c/h2\u003e \u003cp\u003eWild-type and AB strain zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) were maintained at a fish husbandry in IBt, UNAM, Mexico, in a recirculating system at 28\u0026deg;C with a 14 h:10 h light: dark cycle [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Adults were fed daily with newly hatched \u003cem\u003eArtemia nauplii\u003c/em\u003e and a commercial zebrafish diet (Skretting). For breeding, males and females were separated overnight and allowed to spawn the following morning. Eggs were collected approximately 40 minutes after fertilization, washed, and maintained in egg water (60 \u0026micro;g/mL Sea Salt, Instant Ocean) at 28\u0026deg;C until 4 hours post-fertilization (hpf) or sphere stage according to morphological criteria described by Kimmel et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Zebrafish larvae were anesthetized with tricaine (3-aino benzoic acid ethyl ester) solution at a final concentration of 160mg/L in egg water. After completing all experiments, larvae were euthanized by immersion in ice-cold water. All experiments were performed in accordance with relevant guidelines and regulations approved by the Bioethical Committee (Instituto de Biotecnolog\u0026iacute;a, UNAM). Our study is reported in accordance with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Zebrafish embryo exposure and survival assessment\u003c/h2\u003e \u003cp\u003eZebrafish embryo exposure was conducted with slight modifications to the Fish Embryo Acute Toxicity (FET) Test [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Briefly, at the sphere stage, embryos were transferred to 48-well plates (10 embryos per well) containing 300 \u0026micro;L of a 1 mg/mL microplastic suspension in egg water, with one polymer per treatment group. Plates were incubated at 28\u0026deg;C in a humidified chamber. Embryo survival, morphology (compared to that reported by Kimmel et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]), and hatching were monitored daily using a ZEISS Stemi 508 stereomicroscope. Each exposure experiment was carried out in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Touch-evoked response\u003c/h2\u003e \u003cp\u003eTouch-evoked escape responses were assessed at 72, 96, and 120 hpf, following a modified version of Sztal et al.[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Larvae were individually placed in the center of a 10 mm culture dish containing egg water at 28\u0026deg;C, acclimated for 1 minute, and gently stimulated on the trunk with a blunt tip glass probe (up to 10 stimuli per cycle, with 30 seconds rest between cycles). If a larva failed to respond after five cycles, the assay was concluded. A positive escape response was defined as any swimming movement following tactile stimulation. Approximately 30 larvae were analyzed per treatment. Responses were video recorded using a Zeiss Stemi 508 stereomicroscope. Videos were analyzed with manual tracking using Fiji software for the number of stimuli required, swim distance, and active response time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.7 SEM sample preparation: zebrafish larvae\u003c/h2\u003e \u003cp\u003eLarvae at 96 and 120 hpf were fixed in 2.5% glutaraldehyde for 2 hours at 4\u0026deg;C, followed by three 15-minute washes in cold PBS. Post-fixation was performed in osmium tetroxide for 1 hour, followed by three further PBS washes (15 minutes each). Samples were dehydrated on ice using a graded ethanol series (40%\u0026ndash;100%, 10% increments, 15 minutes per step). SEM stubs were cleaned and prepared with carbon adhesive tape during dehydration. Dehydrated larvae were mounted on stubs and gold-coated using an ion sputter coater (10 minutes). Imaging was conducted on a LEICA Stereoscan 440 SEM at 10 kV with preparations stored under vacuum until observation. Images were acquired at 100x, 5000x, 10000x, and 25000x.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.8 Fluorescent staining and confocal imaging\u003c/h2\u003e \u003cp\u003eNeuromasts were stained as described by Owens et al. [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Staining was carried out at 72, 96, and 120 hpf with 0.05% DASPEI (Invitrogen, CA, US) in egg water for 30 minutes, followed by a 1-minute wash and a 5-minute egg water wash. Cell death was detected with 10 \u0026micro;g/mL acridine orange (AO) (Invitrogen, CA, US) for 30 minutes, followed by three 10-minute washes in egg water, modified from Mendieta Serrano et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Bodipy TR (5 \u0026micro;g/mL, Invitrogen) was used for additional staining after AO. All incubations were performed in the dark. Larvae were anesthetized as described previously and embedded in 1.5% low-melting-point agarose and imaged with an Olympus IX81 inverted confocal microscope using 20x and 60x objectives. DASPEI and AO were excited at 488 nm, and Bodipy TR at 543 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eNormality was assessed using the Shapiro-Wilk test. Comparisons between control and each treatment groups were performed using unpaired t-tests were for normally distributed data and Welch\u0026rsquo;s t-tests for non-normal data. Statistical significance was considered at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Analyses were conducted in GraphPad Prism (version 10) with 8\u0026ndash;10 embryos included in each replicate each experiment was conducted in triplicate.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe author(s) declare no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe research was funded by the PAPIIT DGAPA UNAM program IN208122 and IN222325\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eACM: Formal an\u0026aacute;lisis, Investigation, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. NMM: Formal an\u0026aacute;lisis, Methodology Writing \u0026ndash; review \u0026amp; editing. LPL: Writing \u0026ndash; review \u0026amp; editing..TMUR: Methodology, Writing \u0026ndash; review \u0026amp; editing. IHO: Writing \u0026ndash; review \u0026amp; editing. HL:Formal an\u0026aacute;lisis, Supervision Writing \u0026ndash; review \u0026amp; editing. ADVR: Conceptualization, Formal an\u0026aacute;lisis, Supervision, Writing \u0026ndash; review \u0026amp; editing, DSP: ConceptualizationFormal an\u0026aacute;lisis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Enrique Salas and Arlen Ramirez Corona (IBt, UNAM) for their technical support in the acquisition of confocal images. Julio C\u0026eacute;sar Valerio Negreros for technical assistance in confocal microscopy; Dulce Pacheco for help with fish maintenance. To the SECIHTI (CONAHCyT) Ph.D. Scholarship of Cazares Morales (779165) at the Doctorado en Ciencias en la especialidad en Toxicolog\u0026iacute;a at the Centro de Investigaci\u0026oacute;n y Estudios avanzados del Instituto Polit\u0026eacute;cnico Nacional. The research was funded by the PAPIIT DGAPA UNAM program IN208122 and IN222325\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article (and its Supplementary Information files)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJeong, J., Im, J. \u0026amp; Choi, J. Integrating aggregate exposure pathway and adverse outcome pathway for micro/nanoplastics: A review on exposure, toxicokinetics, and toxicity studies. \u003cem\u003eEcotoxicol. Environ. 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A. \u003cem\u003eAn\u0026aacute;lisis del papel funcional de la glutati\u0026oacute;n peroxidasa 4 y de las especies de ox\u0026iacute;geno reactivas en el desarrollo embrionario temprano del pez cebra\u003c/em\u003e (UNAM, 2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"microplastics, zebrafish, neuromasts, toxicity","lastPublishedDoi":"10.21203/rs.3.rs-8398009/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8398009/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroplastic (MP) pollution in aquatic environments is ubiquitous and characterized by particles of highly irregular shapes. Yet, most laboratory studies examining MP impacts on aquatic species rely on pristine polymer spheres, which poorly reflect the diversity and complexity of environmentally derived MPs. In this study, we assessed the developmental effects of environmentally relevant, household-derived, irregular microplastic fragments, on zebrafish. Fragments of synthetised MPs used in this study displayed heterogeneous sizes and jagged shapes, similar to environmental MPs fragments. Acute exposure to all MP types did not induce embryonic lethality or gross malformations but did result in significant sublethal toxicity: exposed larvae showed reduced touch-evoked escape responses, consistent with a pronounced loss or damage of lateral line neuromasts. To further characterize the underlying sensory impairment, we examined neuromast structure and function, which revealed mechanical disruption, kinocilia fusion, and reduced mitochondrial activity. Our findings emphasize that physical and physicochemical interactions associated with fragment morphology and polymer type drive neurosensory toxicity than particle size alone. This work highlights that acute MP exposure disrupts key sensory behaviours and structures critical for ecological fitness. Overall, these results support the need for increased regulatory and scientific attention to behavioural and sensory endpoints in microplastic risk assessments.\u003c/p\u003e","manuscriptTitle":"Behavioural impact of microplastics on zebrafish development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 06:19:09","doi":"10.21203/rs.3.rs-8398009/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-09T06:24:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-06T14:29:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33916200515919280691506147407502267306","date":"2026-03-04T10:26:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"51251935819735048115431118073287008166","date":"2026-03-04T08:34:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71707631123258574829303308926323445267","date":"2026-03-04T08:33:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166426381572550695129086154196318026868","date":"2026-03-04T07:33:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T13:31:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317540915727021231050232683735861094739","date":"2026-02-15T13:20:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55215890202486737171526785744693789075","date":"2026-02-13T13:39:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179928810134876961210688277085631463959","date":"2026-02-13T11:36:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103442754376823214184130486932392891990","date":"2026-02-13T10:24:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-19T00:42:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-29T10:06:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-26T16:06:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-26T15:55:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"041a459b-ad1b-43f2-b796-794a1b3a9997","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":61339260,"name":"Biological sciences/Ecology"},{"id":61339261,"name":"Earth and environmental sciences/Ecology"},{"id":61339262,"name":"Earth and environmental sciences/Environmental sciences"},{"id":61339263,"name":"Biological sciences/Neuroscience"},{"id":61339264,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2026-03-09T06:41:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 06:19:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8398009","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8398009","identity":"rs-8398009","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}