Dynamic Uptake and Elimination of Microplastics by Tetrahymena thermophila

Abstract

Understanding the fate and impacts of microplastics (MPs) on foundational organisms within food webs is essential for accurately assessing their toxicity and environmental risks. While the accumulation and toxicity of MPs in organisms at low trophic levels have been documented, the analysis of the absolute cellular uptake of MPs at a single-cell level with high-throughput methods remains a challenge. In this study, we developed a flow cytometry-based approach integrating a particle balance method and biokinetic modeling to quantitatively assess the uptake and elimination dynamics of fluorescently labeled MPs in the freshwater ciliate Tetrahymena thermophila. The intracellular accumulation of MPs was calculated by subtracting the extracellular MPs from the total exposure amount, ensuring that the measurement was free from interference caused by fluorescence quenching or cellular background noise. Our results revealed that intracellular MPs continuously increased during the first hour, followed by a marked decline within 24 h. By applying a biokinetic model, we demonstrated that the effect of cell division on reducing MP burden is 3.6 to 5.8 times greater than that of elimination, depending on the exposure concentration. The calculated bioconcentration factors (BCFs) exceed 4800, indicating a significant potential for bioaccumulation of MPs in T. thermophila. These results enhance our understanding of the initial processes through which MPs enter the food chain and provide essential technical support for further research on the quantitative toxicity assessment of MP pollution.

1. Introduction

Microplastics (MPs), defined as small plastic particles with a size below 5 mm, are primarily generated unintentionally through the degradation of large plastic materials via physical, chemical, or biological processes in the environment. A subset of the MPs is intentionally manufactured and incorporated into commercial products, including cosmetics and paints. Due to their increasing production and relatively stable chemical properties, MPs demonstrate environmental persistence and have been detected in aquatic, terrestrial, and atmospheric ecosystems. − Aquatic environments, in particular, serve as significant sinks for the MPs, where these particles can be ingested by a wide array of organisms, ranging from planktonic species to larger mammals. , MPs enter the food web through multiple pathways, including adherence, ingestion, and trophic transfer. , Exposure to MPs can lead to various toxicological effects, including growth inhibition, oxidative stress, inflammatory responses, endocrine disruption, malformations, and metabolic alterations. ,

Tetrahymena thermophila, a ciliate protozoan prevalent in freshwater ecosystems, plays an important role in aquatic food webs. Due to its high sensitivity to environmental changes, T. thermophila has been widely used as a model organism for assessing environmental toxicity. − Studies have shown that T. thermophila can ingest and accumulate MPs smaller than 10 μm in natural waters. − The ingestion of MPs disrupts the absorption of essential nutrients in these protozoa, leading to notable reductions in their abundance, biovolume, and carbon biomass. , The MPs also serve as vectors for heavy metals or pollutants, triggering a stress response in ciliates. Moreover, fish larvae readily consume ciliates exposed to MPs than those not exposed, suggesting the potential for the trophic transfer and biomagnification of MPs in aquatic food webs. Therefore, evaluating the bioaccumulation of MPs in T. thermophila is crucial for understanding the ecological implications of MP pollution.

Various analytical techniques have been employed to quantify the accumulation and toxicity of MPs in T. thermophila, including electron microscopy, fluorescence microscopy, and stimulated Raman scattering microscopy. However, these methods are typically low-throughput and are challenging to apply for the quantitative tracking of uptake and elimination kinetics in live T. thermophila due to their rapid motility, which hinders continuous observation. Flow cytometry has emerged as a critical tool for analyzing the accumulation of micro- and nanoplastics at the single-cell level. , In fluorescence-based approaches, MPs are labeled with fluorescent probes, and their intracellular accumulation is inferred from the relative fluorescence intensity within cells. However, quantification remains difficult because intracellular fluorescence can be strongly affected by factors such as dye quenching, fluorescence attenuation, and probe detachment, particularly under the acidic and enzymatically active conditions of lysosomes. − Moreover, cellular autofluorescence and background noise further compromise measurement reliability, making it challenging to establish a reliable calibration relationship between the fluorescence intensity and the number of internalized particles.

The quantification of the uptake and elimination kinetics of MPs in aquatic organisms remains limited due to analytical challenges. Biokinetic models provide a framework for understanding the physiological processes underlying pollutant bioaccumulation, including uptake and elimination rates. − These models have been widely applied to trace metals and nanoparticles in various aquatic organisms, enhancing predictions of bioaccumulation and forming risk assessments. Although previous studies have investigated the ingestion of MPs by ciliates, the biokinetic model that quantitatively describes the behavior of MPs in T. thermophila is limited. Applying biokinetic modeling to T. thermophila can provide detailed insights into the rates of MP uptake and elimination, which are crucial for predicting the fate of MPs within organisms and their potential for trophic transfer in aquatic ecosystems.

In this study, we developed a flow cytometry-based methodology centered on a particle balance (PB) method to address the limitations of fluorescence quantification. The PB approach provides robust quantification through mass balance calculations. By integration of a biokinetic model to quantify uptake and elimination rates, this approach facilitates a comprehensive understanding of MP uptake and elimination within the organism. These results offer valuable insights into the behavior of MPs in aquatic environments and enhance the evaluation of their ecological risks within aquatic food webs.

2. Materials and Methods

2.1. Materials

The fluorescently labeled polystyrene MPs were obtained from Zhichuan Technology Co., Ltd. Proteose peptone and yeast extract were purchased from Becton, Dickinson and OXOID (Thermo Fisher Scientific), respectively. All other chemicals and reagents were of analytical grade and were obtained from commercial suppliers. Ultrapure water (18.2 MΩ·cm) was produced using a Millipore Milli-Q water purification system.

2.2. Characterization of MPs

The morphology of the MPs was characterized via scanning electron microscopy (SEM) using a Quanta FEG 250 instrument (FEI, USA) at an acceleration voltage of 20 kV. The ζ-potential and hydrodynamic diameter of the MPs were measured using a Zetasizer (Nano series, Malvern Instruments, U.K.). The fluorescence emission spectra of the MPs were recorded using a spectrofluorometer (FluoroMax-4, Horiba, USA) to assess their optical properties. Additionally, the stability of the Cy5 dye label was evaluated by incubating the MPs in sterile proteose peptone-based (SPP) medium (2% (w/v) proteose peptone, 0.1% (w/v) yeast extract, 0.2% (w/v) glucose, 0.003% (w/v) ferric citrate, and 1% (v/v) penicillin-streptomycin), PFA solution, and phagolysosomal simulant fluid (PSF) (see Table S1 for composition), respectively, during the typical exposure period.

2.3. Cell Culture and Exposure Methods

T. thermophila strain B2086 was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. Cultures of T. thermophila were performed following the protocols established in our previous research. Briefly, T. thermophila was cultured in SPP medium at 28 °C while being shaken at 135 r/min. Cells in the exponential growth phase were harvested by centrifugation at 800 × g for 5 min, then washed twice in SPP medium, and resuspended in fresh SPP medium to a final density of 1 × 10⁵ cells/mL. This standard cell preparation procedure was applied to the experiments described in Sections and unless otherwise stated.

For the 24 h proliferation assay, cells were exposed to MPs at final concentrations of 0, 10, 20, 50, 100, 200, 500, and 1000 μg/mL. After 24 h of exposure under standard conditions (28 °C and 135 rpm), samples were fixed with 2% PFA for 30 min and stored at 4 °C until analysis.

To investigate the intracellular accumulation of MPs over time (a separate experiment from the proliferation assay), cells were exposed to MPs at final concentrations of 0, 5, 10, 20, 50, and 100 μg/mL under the standard conditions. Samples were harvested at 0.5, 1, 2, 4, 8, and 24 h, fixed with 2% PFA, and stored at 4 °C. Triplicate biological replicates were prepared for each concentration and time point. Prior to analysis, a suspension of MPs alone was analyzed to determine the mean fluorescence intensity of a single particle.

The high range (up to 1000 μg/mL) was used only in the proliferation assay to determine the acute tolerance threshold, a general ecotoxicological practice. − All kinetic studies employed subinhibitory concentrations (5–100 μg/mL) to examine uptake and elimination mechanisms absence of general toxicity.

2.4. Flow Cytometry Analysis

Flow cytometry was employed to analyze the intracellular and extracellular MPs, following the procedures established in our previous work. Prior to analysis, fixed cell samples were vortexed briefly (10 s) to resuspend cells, and 20 μL aliquots were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter, USA). For each event, forward scatter (FSC) and side scatter (SSC) parameters were recorded as the peak height (H) and integrated area (A). The fluorescence signals were collected in the allophycocyanin (APC) channel (excitation: 640 nm, emission: 660/20 nm), recording the fluorescence peak height (APC-H), integrated area (APC-A), and pulse width (APC-W).

For sample analysis, cells were first identified and gated based on FSC-A versus SSC-A. Events outside the cell gate were further examined in APC-A versus SSC-A plots to identify extracellular MPs. The average number of internalized MPs per cell (C _i, particles/cell) was estimated and compared using three independent approaches. The fluorescence peak height (FL-H) method was calculated by dividing the APC-H intensity of MP-containing cells (APC-H_Cell) by the mean APC-H intensity of single MPs (APC-H_MP) (eq ).

$$C_{\mathrm{i}}=\frac{\mathrm{APC}-{\mathrm{H}}_{\mathrm{Cell}}}{\mathrm{APC}-{\mathrm{H}}_{\mathrm{MP}}}$$ 1

The fluorescence area (FL-A) method was calculated similarly but using APC-A values (eq ).

$$C_{\mathrm{i}}=\frac{\mathrm{APC}-{\mathrm{A}}_{\mathrm{Cell}}}{\mathrm{APC}-{\mathrm{A}}_{\mathrm{MP}}}$$ 2

The PB method we employed was a mass-balance framework adapted from previous studies, as defined by eq :

$$C_{\mathrm{i}}=\frac{N_{\mathrm{intra}}}{N_{\mathrm{cell}}}=\frac{N_{\mathrm{expo}}-N_{\mathrm{extra}}}{N_{\mathrm{cell}}}$$ 3

where

$$N_{\mathrm{expo}}=\frac{C_{\mathrm{expo}}\times V_1}{\frac{4}{3}\pi R^3\times \rho }$$ 4

Here, N _intra is the number of MPs in all cells. N _expo represents the total number of MPs introduced into the exposure medium, and N _extra denotes the number of MPs remaining in the medium after exposure, which can be detected by flow cytometry methods. N _cell refers to the number of cells in the exposure medium. C _expo is the exposure concentration of MPs (μg/mL), V ₁ is the total volume of the medium (mL), R is the radius of the MPs (cm), and ρ is the density of polystyrene (1.05 × 10⁶ μg/cm³).

It is important to note that all exposure and kinetic experiments described in this study were performed exclusively with 2 μm MPs. The 2 μm particles were chosen due to their representation of a size fraction that is both commonly found in the environment and exhibit high bioavailability, thereby facilitating efficient ingestion by T. thermophila. To examine the relationship between particle size and pulse width, Cy5-labeled MPs of various sizes (1, 2, 5, 10, and 20 μm) were analyzed. FSC-W and FL-W values were recorded as a proxy for the time a particle remained in the laser beam. For cross-size comparisons, raw values for each particle size were normalized to those obtained for 1 μm MPs, which were set to 1. Data for other sizes were expressed as ratios relative to the 1 μm value under identical instrument settings.

2.5. Confocal Microscopy Analysis

An independent exposure experiment was conducted specifically for confocal microscopy visualization. Following the standard cell preparation procedure (as detailed in Section ), T. thermophila cells were exposed to MPs at a concentration of 100 μg/mL for 0.5, 1, 2, and 24 h. After exposure, cells were fixed with 2% PFA for 30 min, washed twice with PBS, and resuspended before a 20 μL aliquot was placed onto a glass slide for immediate imaging with a laser scanning confocal microscope (Nikon A1 HD25, Japan). Images were processed and analyzed by using ImageJ software (NIH, USA).

2.6. Biokinetic Modeling of MPs Uptake and Elimination in T. thermophila

A distinct experimental design was employed for biokinetic modeling, comprising separate uptake and elimination phases. Cell preparation followed the standard procedure described in Section .

For the uptake phase, cells were exposed to MPs at final concentrations of 0, 5, 10, 20, 50, and 100 μg/mL. Samples were collected at 0.5, 0.75, and 1 h and directly fixed with 2% PFA for 30 min for subsequent flow cytometry analysis.

For the elimination phase, after 1 h of MP exposure, cells were washed twice with fresh SPP medium using mild centrifugation (300 × g for 5 min) to remove extracellular MPs. Control experiments confirmed that this washing procedure removed >99% of free MPs. The washed cells were then resuspended in fresh MP-free SPP medium and continuously cultivated under standard conditions. Samples were collected at 2, 4, 8, 16, and 24 h after resuspension, fixed with 2% PFA, and stored for analysis. During the elimination phase, the intracellular MP count per cell was calculated by accounting for both particle excretion and cell proliferation:

$$C_i=\frac{\mathrm{Total\; MPs\; retained\; intracellularly\; at}t}{N_{\mathrm{cell}}(t)}=\frac{C_{\mathrm{i}}({\mathrm{t}}_0)\times N_{\mathrm{cell}}(t_0)-\mathrm{\Sigma }\Delta N_{\mathrm{excreted}(t)}}{N_{\mathrm{cell}}(t)}$$ 5

C _i(t ₀) is initial intracellular MP count per cell (particles/cell) after 1-h exposure. N _cell(t ₀) is the total cell count at t ₀ (start of elimination phase). ΣΔN _excreted(t) is cumulative MPs excreted into the medium from t ₀ to t, quantified via the flow cytometry detection of extracellular particles. N _cell(t) is the total cell count at time t.

A biokinetic model was adapted to describe the uptake and elimination kinetics of MPs in T. thermophila using the sequential method reported in the organization for economic cooperation and development (OECD) guideline 305. The model is based on a modified first-order kinetic equation:

$$\frac{\mathrm{d}{C}_{\mathrm{i}(t)}}{\mathrm{dt}}=V_{\mathrm{cell}}\times k_1\times {\mathrm{C}}_{\mathrm{w}}(t)-k_2\times C_{\mathrm{i}(t)}$$ 6

$$C_{\mathrm{i}(\mathrm{t})}=V_{\mathrm{cell}}\times C_{\mathrm{w}}\times \frac{k_1}{k_2}\times (1-{\mathrm{e}}^{-k_2\times t})+{\mathrm{C}}_{\mathrm{i}(t_0)}\times {\mathrm{e}}^{-k_2\times t}$$ 7

Where C _i and C _w represent the concentration of MPs in cells (particles/cell) and medium (particle/mL). V _cell is the cell volume, which is taken as 7.04 × 10^–9 cm³ based on Werlin et al. k ₁ and k ₂ are the uptake rate constant (h^–1) and elimination rate constant (h^–1). As T. thermophila are fast-growing organisms, with a generation time of approximately 2–4 h under optimal culture conditions, the growth rate should be included in the elimination rate constant. After T. thermophila is transferred to fresh SPP medium, the initial concentration of MPs in the medium is zero (C_w(0) = 0). For modeling the elimination kinetics, we assume that the subsequent concentration of excreted MPs remains low enough that reuptake is negligible. Under these conditions, the concentration of MPs in T. thermophila during elimination can be described as follows:

$$C_{\mathrm{i}(t)}=C_{\mathrm{i}(t_0)}\times {\mathrm{e}}^{-k_2\times t}=C_{\mathrm{i}(t_0)}\times {\mathrm{e}}^{-(k_{\mathrm{e}}+\mu )\times t}$$ 8

Where the elimination rate constant k ₂ comprises two components: k _e is the “true” elimination rate constant and μ is the growth dilution rate constant. This formulation reflects the combined effects of elimination and organismal growth on the reduction of MP concentrations within cells. These kinetic parameters are derived under general model assumptions, including a constant growth rate during the elimination phase, which is justified by the stable exponential growth of cell nutrient-rich medium, and a homogeneous exposure across the population. , These assumptions, together with first-order uptake and elimination kinetics, , yield robust population-average parameters derived from our flow cytometry data encompassing over 2000 cells per sample.

The bioconcentration factor (BCF) was calculated as

$$\mathrm{BCF}=\frac{k_1}{k_2}$$ 9

This factor serves as an indicator of the potential for MPs accumulation within T. thermophila. A higher BCF suggests a greater bioaccumulation potential.

2.7. Data Analysis

Flow cytometry data were analyzed using FlowJo software (version 10.0, FlowJo LLC, USA), while SEM and confocal microscopy images were processed using ImageJ (version 1.52a, NIH, USA). Statistical analyses were performed using SPSS software (ver. 22.0, IBM Corp., USA). All experiments were conducted with triplicate biological replicates (n = 3), and data are presented as mean ± standard deviation (SD). Differences among groups were evaluated by using one-way analysis of variance (ANOVA). For statistically significant ANOVA results, posthoc multiple comparisons were performed using Tukey’s Honestly Significant Difference (HSD) test. The effect size for the ANOVA was calculated as eta-squared (η²), and for pairwise comparisons, Cohen’s d was reported. A significance level of p < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Characterization of Fluorescently Labeled MPs

As shown in Figure A, the fluorescently labeled MPs displayed a spherical morphology with an average size of 2.02 ± 0.13 μm. To assess colloidal stability, hydrodynamic diameters and zeta potentials of MPs in water, SPP medium, PFA solution, and PSF solution were measured by DLS (Figure B, C). Hydrodynamic diameters of the fluorescently labeled MPs were around 2.3 ± 0.3 μm across all media. The zeta potentials of MPs were approximately −20 mV in water, PFA solution, and PSF solution. In the SPP medium, it increased to −10 mV. These results confirm that the MPs used in this study exhibit a uniform particle size with high stability.

1.

Characterizations of fluorescently labeled MPs. (A) SEM image and size distribution. (B) Hydrodynamic diameter and (C) zeta potentials of MPs in water, PFA, SPP, and PSF medium, respectively. (D) Fluorescence emission spectra of MPs with an excitation wavelength of 635 nm. (E) Representative fluorescence images after 24 h of incubation in test solutions. (F) Normalized fluorescence intensity of single MPs. (G) Linear correlation between flow cytometry counts and MP concentration. (H) Relative MPs percentage in different solutions during 24 h.

A sustained and robust fluorescent stability of labeled MPs is imperative for an accurate examination of the accumulation and kinetics of MPs within intricate biological matrices. As depicted in Figure D, the fluorescence analysis revealed a maximum emission peak at approximately 668 nm when excited at a wavelength of 635 nm, exhibiting a fluorescence spectrum comparable to that of Cy5. The fluorescence intensities of the MPs remained stable after 24 h of incubation at 28 °C in SPP, PFA, and PSF (Figure E and F). We further explored the stability of MPs by flow cytometry. As shown in Figure G, the counting results revealed a strong linear correlation between the number of MP particles and their concentrations. Crucially, the particle numbers in various media remained largely unchanged in cell-free systems, indicating that nonbiological loss of MPs was negligible (Figure H). This ensures that the depletion of particles from the exposure medium in the presence of cells can be reliably attributed to biological uptake. The stability profile, characterized by the consistent fluorescence intensity and particle count over the experimental period, alongside the direct quantification of dye leakage, demonstrating less than 0.7% release across all tested media (Figure S1), provides compelling evidence that the covalent linkage of the Cy5 dye to the polystyrene matrix is highly stable. This minimal leaching ensures that subsequent flow cytometric measurements and observed biological responses are exclusively attributable to the particulate MPs rather than artifacts from free fluorescent dye.

3.2. Flow Cytometric Detection and Quantification of MPs in T. thermophila

Flow cytometry was employed to analyze the accumulation of MPs in T. thermophila using a novel gating strategy. After incubated with fluorescent MPs, T. thermophila were fixed with PFA and the mixture was introduced into a flow cytometer without further treatment. As shown in Figure A, single T. thermophila cells and extracellular MP particles were effectively distinguished based on the FSC, SSC parameters, and Cy5 signals. The size disparity between polystyrene MPs (approximately 2 μm) and T. thermophila cells (20–40 μm) allowed for clear separation of the two populations, while the fluorescence intensity of the Cy5 channel was used to assess the amount of extracellular MPs.

2.

(A) Gating strategies for identifying T. thermophila cells, cell debris, and extracellular based on SSC and FSC. (B) Cell counts of T. thermophila after exposure to different concentrations of MPs for 24 h (n = 3). Significance versus control was assessed by one-way ANOVA with Tukey’s HSD test (*p < 0.05). Cohen’s d for these comparisons indicated large effect sizes (1.87–5.51). (C) Representative gating strategies for extracellular MPs at 0.5 h. (D) Time-dependent changes in the percentage of extracellular MPs at different exposure concentrations. (E) Comparison of particle balance (PB) quantification, fluorescence height (FL-H) quantification, and fluorescence area (FL-A) quantification methods after 1 h of exposure. (F) Time-dependent changes in the number of intracellular MPs at different exposure concentrations.

First, we investigated the impact of MPs on the growth of T. thermophila. As shown in Figure B, MPs exhibited a dose-dependent effect on the density of T. thermophila. Concentration up to 200 μg/mL had no significant effect on the density of T. thermophila after 24 h. However, at higher concentrations, the cell density of T. thermophila decreased as MP concentration increased, with decreases of 10.68 and 20.23% at 500 and 1000 μg/mL, respectively. To definitively attribute the observed toxicity to MPs rather than potential dye leakage, we conducted a comprehensive exposure experiment comparing bare MPs, Cy5-labeled MPs, and the leachate from incubated MPs. As shown in Figure S2, both bare and Cy5-labeled MPs caused significant and comparable growth inhibition, while the leachate containing the maximum potential free dye showed no toxicity. This unequivocally demonstrates that the antiproliferative effects originate from the particulate MPs themselves rather than any fluorescent dye artifacts. Subsequently, the accumulation processes of MPs in T. thermophila were examined at concentrations lower than 100 μg/mL, which did not significantly affect cell growth.

Using the gating strategy described above, the extracellular MPs were gated after exposure for 0.5 h (Figure C). The number of extracellular particles increased with the increased exposure concentrations. By calculating the extracellular MPs over time, we found that the proportion of extracellular MPs relative to total MPs gradually decreased (Figures D and S3). This implies continuous uptake by the cell populations throughout the duration of the experiment. The decreasing percentage of extracellular MPs directly reflects the time-dependent decrease in the extracellular concentration, which drives the uptake process. The absence of significant particle loss in cell-free controls (Figure H) confirms that this decrease is due to biological ingestion and not experimental artifact. Meanwhile, the mean fluorescence intensity (MFI) in T. thermophila populations was obtained (Figure S4), suggesting a dose-dependent accumulation of MPs within these cells. The intracellular MPs per cell were calculated by traditional FL-A and FL-H methods, as shown in Figure E. Interestingly, the particle numbers measured by the FL-A method were substantially higher than those obtained with the FL-H method. Furthermore, we utilized PB method to calculate intracellular MPs by subtracting the number of extracellular MPs from the total input and normalizing to cell counts. The results showed that the particle numbers calculated by the PB method were consistent with those obtained from the FL-H analysis (Figure E). The intracellular particle numbers obtained using FL-H and PB methods were more reliable than those from FL-A method. First, the calculated intracellular particle number by FL-A method was more than 1350 per cell at 100 μg/mL, whereas confocal microscopy did not reveal such dense intracellular accumulation. Besides, such a particle number was higher than the total particle number (2.27 × 10⁷ particles/mL) added to the system. Finally, the PB method, grounded in this mass-balance principle and independent of fluorescence artifacts, yielded physically plausible results. Therefore, we further verified this using confocal microscopy and employed the reliable PB method for subsequent analysis.

Finally, we utilized the PB method to calculate the time-dependent accumulation of MPs by T. thermophila at different exposure concentrations. As shown in Figure F, the number of particles within T. thermophila cells increased rapidly in the first hour. However, with prolonged exposure, a subsequent decline in particle numbers was observed, indicating a reduction in the single-cell intracellular burden of MPs. This decline indicates the presence of mechanisms that mitigate MP accumulation, such as dilution through cell division and active excretion. This same nonmonotonic trend was also observed in the FLA time-series data (Figure S4), indicating it is not an artifact of the PB method. Consequently, we proposed a highly complex process involving uptake, elimination mechanisms, and growth dilution during the accumulation of MPs by T. thermophila.

3.3. Laser Scanning Confocal Microscope to Visualize the MPs Accumulation

To validate the accumulation processes of MPs identified through flow cytometry, confocal microscopy was utilized to visualize the uptake and distribution of MPs in T. thermophila cells. An overview of the entire cell population shown in Figure S5, whereas Figure A presents high magnification images of individual cells. The results indicated that T. thermophila cells exhibit a time-dependent accumulation of MPs after exposure to 100 μg/mL MPs. SEM imaging (Figure S6) confirmed that no MPs adhered to the cell surface, likely due to the considerable size disparity between the cells and the MPs, as well as the presence of cilia on the cell surface.

3.

Confocal microscopy and quantitative analysis of MPs uptake in T. thermophila. (A) Representative confocal images of T. thermophila cells exposed to 100 μg/mL MPs: (1) bright field (BF) image, (2) MPs fluorescence image, and (3) merged images of (1) and (2). Scale bar = 30 μm. (B) Quantification of the average number of microparticles internalized per cell following exposure to 100 μg/mL MPs.

At 0.5 h postexposure, MPs were predominantly localized near the oral apparatus of T. thermophila. As the exposure time increased, MPs gradually accumulated near the cytoproct, indicating that T. thermophila may uptake MPs through its oral apparatus and subsequently process them via the cytoproct, which can be observed in Supplementary Video 1. These observations are consistent with the known feeding and digestion mechanisms of T. thermophila, wherein particles are ingested via the oral apparatus and transported through the cytopharyngeal vesicles before being expelled through the cytoproct. ,

To independently validate the temporal dynamics of MP accumulation, the number of particles per cell within the confocal microscope field was determined, as shown in Figure B. The number of particles per cell obtained via confocal microscopy was lower than that obtained by the PB method at each time point. This discrepancy can be explained by the distinct sensitivities and technical principles inherent to each analytical approach. Both methods revealed highly consistent temporal dynamics characterized by a rapid uptake phase followed by a pronounced clearance phase. Similar behaviors have been observed in other protozoa exposed to nanoparticles, , suggesting a potential regulatory mechanism governing particle accumulation in response to exposure. The convergence of results from these two complementary analytical approaches provided strong support for the validity of the described dynamic process.

3.4. Dynamic Uptake and Elimination of MPs by T. thermophila

To further interpret the uptake and elimination trends described above, we quantitatively modeled MPs dynamics in T. thermophila using a biokinetic approach (Figure A). The model described the rates of MPs uptake and elimination in T. thermophila under different exposure concentrations. The model showed an excellent fit to the experimental data (Figure B), and the calculated parameters for each concentration are summarized in Table . k ₁ increased with increasing MP concentrations, suggesting an increase in the efficiency of MP uptake at higher concentrations. The increase in k ₁ may be attributed to the enhanced contact frequency between cells and MPs due to higher particle availability in the medium. Similarly, k _e also increased with higher concentrations, reflecting the cell’s enhanced ability to expel MPs as the exposure concentration increased, the changing trend of k _e is consistent with that reported in the literature. ,

4.

Dynamics of MPs accumulation in T. thermophila. (A) Experimental design for MPs uptake and elimination in T. thermophila. (B) Uptake and elimination kinetics showing experimental data (symbols, n = 3) and the fitted biokinetic model (solid lines). (C) Change in the ratio of μ/k _e and change in the BCF at different concentrations. (D) Mechanistic summary of MPs biokinetics in T. thermophila.

1. Biokinetic Model Parameters for MP Uptake and Elimination in T. thermophila at Different Exposure Concentrations.

MPs con. (μg/mL)	k 1 (h–1)	k 2 (h–1)	μ (h–1)	k e (h–1)
5	944.201 ± 38.203	0.195 ± 0.019	0.166 ± 0.007	0.029 ± 0.020
10	987.775 ± 34.054	0.201 ± 0.017	0.168 ± 0.008	0.033 ± 0.019
20	1063.683 ± 39.707	0.213 ± 0.020	0.171 ± 0.009	0.042 ± 0.022
50	1116.952 ± 35.194	0.223 ± 0.017	0.175 ± 0.008	0.048 ± 0.019
100	1089.358 ± 41.495	0.208 ± 0.020	0.163 ± 0.006	0.045 ± 0.021

Meanwhile, we calculated the μ/k _e ratio within the biokinetic model to assess the relative contributions of growth dilution and elimination to reducing intracellular MPs. As shown in Figure C, the μ/k _e ratios were larger than 1 at all concentrations, indicating that growth dilution is the primary mechanism for reducing the intracellular MPs burden rather than elimination processes. At low MPs concentrations, 5 μg/mL for example, the μ/k _e ratio is as high as 5.802, suggesting that the reduction of intracellular MPs is primarily the result of dilution due to cell division. As the exposure concentration increased to 100 μg/mL, the ratio decreased to 3.631. Correspondingly, a high exposure concentration increased the contribution of elimination to the reduced content of intracellular MPs, but it was still smaller than that of cell division (Figure D). The BCF is defined as the ratio of the concentration of a chemical in an organism to that in the environment, and serves as a key indicator of bioaccumulation potential. , In this study, the BCFs of MPs in T. thermophila consistently exceeded 4800 across all exposure concentrations (Figure C), a level that qualifies as high bioaccumulation according to established international criteria such as the European Union’s regulation on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) and the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS). − This pronounced bioconcentration can be mechanistically attributed to the extremely high uptake rate constant observed in our biokinetic model coupled with an elimination process dominated by growth dilution rather than active excretion. Ecologically, these findings underscore the role of T. thermophila as a significant vector for MPs in aquatic systems. While growth dilution moderates the intracellular MP burden at the individual level, it concurrently promotes the propagation and dispersal of MPs through continuous biomass production, thereby potentially amplifying the risk of trophic transfer at the base of aquatic food webs. Consequently, the high BCF values highlight the considerable potential for MPs to enter and be transferred within aquatic food chains via protozoan intermediaries.

Investigating the accumulations and impacts of MPs in protozoan ciliates is essential for understanding their environmental behavior and bioaccumulation across trophic levels. In recent years, flow cytometry has served as a powerful tool for the detection of MPs in water samples. − It directly quantifies accumulation of fluorescent dye-labeled MPs in ciliate cells by converting fluorescence intensities to the number of ingested particles. The intracellular MPs are calculated by the FL-A and FL-H methods from the cellular fluorescence intensity in flow cytometry. However, a significant discrepancy was observed between the results generated by the two methodological approaches in our investigation, especially at high concentrations. This was primarily attributed to the size-dependent fluorescent signals as shown in Figure .

5.

Size- and concentration-dependent pulse width effects on flow cytometry quantification. (A) FSC-W of MPs with different diameters and T. thermophila, showing longer FSC-W for larger particles due to extended transit times in flow cytometry. (B) FL-W of MPs with different diameters, exhibiting more pronounced size-related differences compared to FSC-W. (C) FL-W of T. thermophila exposed to different concentrations of MPs, showing increased FL-W with higher MP concentrations.

To examine the relationship between particle size and pulse width, Cy5 labeled MPs of various sizes (1, 2, 5, 10, and 20 μm) were analyzed (Figure S8). For large particles, the pulse FSC-W was larger than that of small particles due to the longer transit time through the laser (Figure A). The pulse width of FL-W also increased with the particle size (Figure B). As a result, the FL-A obtained from FL-W and FL-H was higher for large particles than that of small particles, leading to an overestimation in the particle number determination for large particles. Likewise, the FL-W in T. thermophila sharply increased with MPs concentrations (Figure C), probably due to the aggregation of MPs in the cells. The FL-A was much higher than FL-H when the MPs concentrations increased (Figure E). On the other hand, FL-H can be influenced by fluorophore stability, quenching, instrument settings, and background interference. In contrast, the proposed PB method was independent of signal distortions from cell size, internal complexity, or pulse shape artifacts, providing a novel strategy to estimate the intracellular MPs. Furthermore, the PB method was inherently robust against potential artifacts arising from dye leaching. This was attributed to the gating strategy that extracellular MPs were defined by both the light-scattering and fluorescent profiles, which minimized the interference of free dye molecules. Consequently, the calculation of internalized particles via mass balance remains unaffected by the presence of free or internalized dye, as the method does not rely on fluorescence intensity correlations. It simplifies the experimental procedures and is particularly suitable for heterogeneous systems where cells and particles differ vastly in size.

Moreover, the biokinetic model provides a comprehensive framework for understanding the interactions between T. thermophila and MPs at varying concentrations. Our results demonstrate that both uptake and elimination rates are positively correlated with the initial exposure concentration of MPs. The model underscores the predominant role of cell division in MPs clearance while revealing the increasing importance of active expulsion at higher MPs concentrations. This nuanced perspective is crucial for evaluating the ecological risks associated with MPs pollution, particularly concerning trophic transfer and accumulation in aquatic ecosystems. Previous studies reported similar uptake and elimination dynamics for the bioaccumulation of trace metals and nanoparticles in microorganisms, where growth dilution plays a significant role in elimination. , However, the kinetic parameters that we used are significantly distinct from those reported for other contaminants and organisms. For instance, the uptake rate constants for MPs in T. thermophila are higher than those reported for trace metals in marine invertebrates, suggesting a more efficient uptake mechanism for particulate contaminants in protozoa. Conversely, the elimination rate constants observed in our study are lower than those reported for graphene nanoplatelets and carbon nanotubes, which may reflect the unique physical properties of MPs and the limited pathways available for their excretion. These differences underscore the importance of contaminant-specific kinetic studies to accurately predict bioaccumulation patterns and provide critical insights into the environmental behavior of diverse pollutants.

Additionally, given the high BCF observed for MPs, further research is needed to explore the long-term effects of MP exposure on T. thermophila. Potential impacts on reproductive success, gene expression related to stress responses, and interactions with other environmental factors, such as co-occurring pollutants, should be investigated. Besides, the fluorescent labeling itself may introduce interference effects, potentially altering either the organisms’ physiological responses or the accuracy of MP interaction detection. Addressing these research gaps is critical as it will enable a more robust assessment of the broader ecological risks posed by MPs and inform the development of targeted strategies to mitigate their impacts on aquatic ecosystems.

This study introduces a mass-balance-based quantitative method for particulate matter, combined with a biokinetic model, using T. thermophila as a model organism to investigate the uptake and elimination kinetics of MPs in unicellular organisms. This approach addresses the limitations of fluorescence-based detection methods, avoiding signal bias caused by the intracellular environment. The study found that T. thermophila primarily reduced its MP load through growth dilution rather than through active excretion. While this mechanism may mitigate toxicity at the individual level, it simultaneously promotes the dissemination of MPs by acting as a biological vector at the base of the aquatic food web. By revealing extremely high bioconcentration factors and ecological transmission pathways, this research provides methodological innovations and mechanistic insights for assessing the ecological risks of MPs.

4. Conclusions

In this study, we established a flow cytometry-based strategy to quantify the uptake and elimination dynamics of MPs in T. thermophila, enabling clear separation of intracellular and extracellular particles and integration of cell growth effects into kinetic modeling. The tolerance concentration of T. thermophila to MPs (2 μm) was 200 μg/mL, with concentrations above this threshold inhibiting cell growth rates, indicating that the high tolerance of T. thermophila to MPs may exacerbate the accumulation of MPs in the food chain. The extracellular MPs were effectively distinguished from intracellular MPs and debris based on scatter and fluorescent signals. At exposure concentrations below 200 μg/mL, the intracellular MPs contents increased during the first hour postexposure, followed by a sharp decrease within 24 h. In contrast, the extracellular MPs contents continuously decreased throughout the experiment. We further calculated the uptake and elimination rate using a modified first-order kinetic equation, incorporating the growth rates of T. thermophila into the elimination rate constant to account for cell division effects. Both uptake and elimination rates are positively correlated with the initial exposure concentrations of MPs. Using the BK model, we found that the contribution of cell division to reducing MP burden overweighs that of elimination by 3.6 to 5.8 times, depending on the exposure concentrations. Although higher exposure concentrations enhanced the role of elimination, it remained less significant than cell division. The BCFs for MPs exceeded 4800, indicating a high potential for bioconcentration that could lead to significant bioaccumulation. Our studies provide a strategy to investigate the interaction dynamics and bioconcentration effects of MPs on aquatic foundational organisms. These findings enhance our understanding of the initial processes by which MPs enter the food web and highlight the need for further research on the long-term ecological impacts of MPs pollution.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.5c00435.

• Additional materials and methods, figures, tables (PDF)

• Supplementary video (AVI)

Yaquan Liu: Writing – original draft, Investigation, Formal analysis, Methodology, Visualization. Jie Gao: Conceptualization, Writing – review and editing, Project administration, Supervision, Funding acquisition. Junjie Ma: Investigation. Liu Zhang: Investigation. Qi Wu: Investigation. Xinyue Yang: Investigation. Ziniu Wang: Investigation. Gang Tang: Investigation. Haijiang Tian: Investigation. Yun Ding: Investigation. Guangxuan Wang: Investigation. Runzeng Liu: Supervision. Li Zeng: Conceptualization, Writing – review and editing, Project administration, Supervision, Funding acquisition. Guangbo Qu: Conceptualization, Writing – review and editing, Project administration, Supervision, Funding acquisition. Guibin Jiang: Supervision, Funding acquisition.

The authors declare no competing financial interest.