Technical Review: Toxic effects of biodegradable polylactic acid nanoplastics on developing zebrafish (Danio rerio)
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Authors: F. Scalia, F. Capparucci, M. D. Amico, M. Marino, E. P. Lamparelli, L. Longhitano, S. Giallongo, R. Falleti, F. Rappa, C. Iaria, F. Marino, G. Della Porta, A. Marino Gammazza, F. Bucchieri, A. Santoro, F. Cappello, and M. A. Szychlinska
Affiliations: University of Salerno, Baronissi, Italy
Background Introduction
While biodegradable plastics have been promoted as environmentally sustainable alternatives to petroleum-based polymers, emerging evidence suggests they may pose comparable toxicological risks. Polylactic acid (PLA) represents approximately 33% of global bioplastic production and is widely utilized in agriculture, medical devices, and food packaging. However, PLA disposal in aquatic environments exposes it to suboptimal degradation conditions. This leads to fragmentation into environmentally persistent micro- and nanoplastics, challenging the assumption that biodegradability equals reduced environmental impact.
To assess potential impacts, the zebrafish (Danio rerio) model organism was selected due to its well-established utility in developmental and environmental toxicology, featuring 70% genetic homology with humans, rapid embryonic development, optical transparency for real-time visualization of internal tissues, and capacity for high-throughput screening. These fish were then used for a comparative study between PLA and polystyrene (PS), a reference petroleum-based polymer. This study directly addresses a significant knowledge gap: while PS microplastics have been extensively studied in zebrafish, investigations of PLA-derived nanoplastics remain limited, with only one prior study examining PLA-NPs of 80 nm diameter.
Materials and Methodology
The researchers produced PLA nanoparticles (PLA-NPs) with microfluidics-assisted nanoprecipitation by mixing PLA dissolved in acetone with an aqueous polyvinyl alcohol solution. These PLA-NPs incorporated rhodamine B as a fluorescent tracer and were dosed to wild-type zebrafish embryos. Embryos were subjected to toxicity assessment, gene expression analysis, and histological analysis at intervals from 24 to 120 hours post-fertilization (hpf). Additional uptake testing was also conducted on human dermal fibroblasts (HDFs) to assess human skin barrier penetration. PS microparticles (PS-MPs) were used as a reference point throughout these tests.
Results
Bioaccumulation Kinetics and Spatial Distribution:
Both PLA-NPs and PS-MPs demonstrated concentration-dependent bioaccumulation in developing zebrafish in a temporal progression. Prior to hatching (0-48 hpf), significant fluorescence was observed around the chorion at 1 mg/L exposure. Following hatching, particles adhered to larval skin surfaces, particularly at elevated concentrations. At the protruding-mouth stage (72 hpf), the yolk sac and digestive tract emerged as primary accumulation sites. By 96-120 hpf, both particle types localized predominantly to the gastrointestinal tract in a dose-dependent manner. Crucially, at 120 hpf, PLA-NPs accumulated as large aggregates in the anterior intestinal bulb, appearing to create an obstructive mass that impeded alimentary canal transit—a phenomenon not observed with PS-MPs. The fluorescence intensity of PLA-NPs increased in a time- and dose-dependent manner through 120 hpf, whereas PS-MPs fluorescence declined at this endpoint, suggesting differential elimination kinetics. This differential bioaccumulation pattern indicates that the smaller size and distinct surface properties of PLA-NPs promote aggregation and retention, potentially leading to prolonged exposure and enhanced tissue-level effects compared to larger PS-MPs.
Physiological Effects on Cardiac Function:
Standard ZFET parameters (mortality, hatching rate, body length, visible developmental malformations) revealed no significant alterations between control and treatment groups at either concentration. However, significant alterations in heartbeat rate emerged as a critical finding, with opposing directional effects between particle types. PLA-NP-exposed larvae demonstrated significant bradycardia (decreased heart rate) at 96 and 120 hpf at the 1 mg/L concentration, with two individual larvae exhibiting transient cardiac arrest followed by resumption of beating, suggesting potential myocardial depression or electrical conduction abnormalities. Conversely, PS-MP exposure induced significant tachycardia (increased heart rate) at both concentrations and timepoints. The PLA-NP-induced bradycardia is mechanistically significant, as it may result from lactic acid accumulation during PLA nanoplastic degradation, lowering intracellular pH and triggering inflammatory responses and metabolic dysfunction similar to mechanisms observed in medical implant degradation and lactic acidosis pathophysiology. The opposing cardiac responses between PLA-NPs and PS-MPs, despite similar bioaccumulation patterns, indicate distinct physicochemical and degradation characteristics governing their physiological impacts.
Oxidative Stress and Antioxidant System Dysregulation:
Gene expression analysis revealed distinct patterns of oxidative stress and adaptive responses between treatment groups. In PLA-NP-exposed larvae, all oxidative stress markers (hmox1a, sod1, sod2, nos2) showed significant upregulation at both 72 and 120 hpf, indicating sustained acute oxidative stress and robust antioxidant system activation. This consistent upregulation suggests that PLA-NPs induce acute, potentially reversible oxidative stress that triggers compensatory antioxidant responses. In contrast, PS-MP-exposed larvae displayed dysregulated antioxidant responses: hmox1a and nos2 were markedly upregulated at 72 hpf but significantly downregulated at 120 hpf, while sod1 remained downregulated at 120 hpf despite earlier upregulation. This temporal remodeling pattern indicates progressive disruption of redox balance, evolving from acute stress response to chronic oxidative dysfunction characterized by antioxidant system exhaustion and net accumulation of reactive oxygen species. The divergent oxidative stress responses highlight that despite comparable bioaccumulation, PLA-NPs and PS-MPs engage fundamentally different cellular stress mechanisms, with PLA-NPs promoting acute reversible stress and PS-MPs promoting chronic irreversible oxidative dysfunction.
Inflammatory and Immune Response Dysregulation:
PLA-NP exposure induced significant upregulation of pro-inflammatory markers (il1, tnf, ifn-γ, il13) predominantly at 120 hpf and at the 1 mg/L concentration, suggesting acute inflammation progression during later developmental stages. The anti-inflammatory cytokines il4 and il13 were upregulated at the early 72 hpf timepoint but downregulated at 120 hpf, implying an initial immune dampening followed by inflammatory dominance. Notably, tbx21 (T-box transcription factor 21, a marker of type 1 immune response) showed significant upregulation at both 72 and 120 hpf, indicating sustained Th1-type immune activation. PS-MP exposure elicited a distinct immune dysregulation pattern: pro-inflammatory markers (il4, ifn-γ, tnf, tbx21) were induced at 72 hpf but suppressed at 120 hpf, with only il1 remaining upregulated at the highest concentration at the later timepoint. This temporal dissociation suggests that PS-MPs trigger early immune activation followed by immunosuppression, consistent with chronic oxidative stress-induced immune dysfunction. The inflammation findings are significant because they occur despite larvae lacking adaptive immunity at this developmental stage, indicating that nanoplastic effects occur through innate immune pathways. The selective induction of type 1 (Th1) immune responses suggests potential immunotoxic skewing that could compromise host defense capacity.
Histological Analysis and Tissue Morphology:
Hematoxylin and eosin staining revealed no visible alterations in brain and liver tissue of PLA-NP-exposed larvae at either concentration. Conversely, PS-MP-exposed larvae demonstrated significant morphological pathology: both low (0.1 mg/L) and high (1 mg/L) PS-MP concentrations induced widespread vacuolization manifesting as spherical empty spaces creating tissue discontinuity in both brain and liver. The absence of detectable vacuolization in PLA-NP-treated tissue despite strong oxidative stress and inflammatory gene expression indicates that oxidative damage at these concentrations may be restricted to subcellular (potentially nuclear) levels rather than gross tissue architecture. This finding suggests that PLA-NPs may cause early molecular pathology without manifesting as gross morphological damage at this developmental stage, implying that proteomic and genomic alterations precede tissue-level structural changes. The vacuolization in PS-MP-exposed tissues correlates with hepatomitochondrial dysfunction and is associated with oxidative stress-driven autophagy and cell death processes.
Human Dermal Fibroblast (HDF) Uptake and Cellular Internalization:
Flow cytometry analysis quantified differential cellular uptake kinetics between particle types. PLA-NPs demonstrated concentration-dependent uptake, reaching only 6% positive cells at the highest concentration (300 μg/mL) at 24 hours, maintained through 72 hours. In contrast, PS-MPs showed superior cellular internalization, achieving 33% positive cells at 300 μg/mL at 24 hours, increasing to 45% by 72 hours. Despite the lower absolute uptake rate, PLA-NP internalization in HDF cells is significant because it demonstrates that nanoplastics can penetrate the human dermal barrier, a critical finding for understanding transdermal exposure routes. Immunofluorescence confocal microscopy revealed that internalized PLA-NPs accumulated in the cytoplasmic region with preferential concentration in perinuclear space, similar to PS-MP distribution patterns. The authors hypothesize that the lower HDF uptake of PLA-NPs compared to PS-MPs may reflect aggregation of PLA-NPs before reaching the cell membrane, reducing bioavailable nanoparticle numbers. Alternatively, the distinct surface chemistry and physicochemical properties of PLA versus PS polymers may influence membrane interaction and transport efficiency. Critically, this finding demonstrates that biodegradable PLA-NPs, despite being touted as safer alternatives to conventional plastics, can traverse biological barriers and accumulate intracellularly in human cells.
Conclusion
Scalia and colleagues established that biodegradable PLA nanoplastics, despite degrading more readily than petroleum-based polymers, bioaccumulate within developing zebrafish at environmentally relevant concentrations and induce significant cellular stress responses, physiological alterations, and immunotoxic effects. The study challenges the prevailing assumption that biodegradability guarantees reduced ecotoxicological risk. PLA-NPs uniquely induced cardiac bradycardia and sustained oxidative stress responses, distinct from PS-MP-induced tachycardia and chronic oxidative dysfunction. Critically, both PLA-NPs and PS-MPs demonstrated capacity to penetrate human dermal fibroblasts, establishing a concerning precedent for potential human health impacts through dermal and gastrointestinal routes.
This paper utilized the PreciGenome NanoGenerator Flex-M for PLA-NP synthesis. While pressure-based microfluidics is most often used for lipid nanoparticle (LNP) synthesis, it can also be effective for polymer NPs due to tunable starting conditions and uniform mixing.
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