# Adsorption Behavior of Nonylphenol on Polystyrene Microplastics and Their Cytotoxicity in Human Caco-2 Cells

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Materials and Chemicals

#### 2.2. Characterization of PS-MPs and Detection of NP

_{2}adsorption-desorption using Brunauer-Emmett-Teller (JW-BK132F, JWGB Sci & Tech Ltd., Beijing, China). The surface functional groups of the PS-MPs were measured usin Fourier transform infrared spectroscopy (FTIR, Nicolet iS50, Thermo Fisher Scientific, Waltham, MA, USA) in the wavenumber range of 4000–400 cm

^{−1}with KBr pellet.

#### 2.3. Adsorption and Desorption Experiments

_{e}) at equilibrium (t = 96 h) or a selected time (q

_{t}) were calculated, respectively, by Equations (1) and (2):

_{0}, C

_{e}, and C

_{t}are the solution concentration of NP at initial, equilibrium (t = 96 h), and selected times t (h), respectively; V (mL) is the volume of solution and m (g) represents the weight of the PS-MPs.

_{dt}(mg/g) denoted the desorption capacity of the PS-MPs at the time t (h); C

_{d0}and C

_{dt}are the concentration of NP in the solution at an initial and selected time t (h); V

_{d}(L) was the volume of solution added, and m

_{d}(g) was the total weight of the PS-MPs and adsorbed NP used for the desorption experiment.

_{dt}, %) can be calculated based on Equation (4):

#### 2.4. The Factors That Influence the Adsorption Behavior of NP on PS-MPs

^{+}, Fe

^{2+}, Ca

^{2+}, and K

^{+}respectively) until the equilibrium of the NP solutions (4 mg/L) and the 0.1 μm PS-MPs (20 μg/mL) was established. In addition to the normal influencing factors, the initial concentration of NP and the particles sizes of the PS-MPs are non-negligible effects. The influence of the initial concentration of NP was investigated by agitating the NP solution at a series of gradients (1, 4, 10, 20, 30, and 40 mg/L) with 20 μg/mL 0.1 μm PS-MPs at 25 °C for 96 h. The effect of the diameter of the PS-MPs on NP adsorption was carried out in NP solutions (4 mg/L) with 20 μg/mL PS-MPs, with different particle sizes (0.1, 1, 10, 50, and 100 μm) at 25 °C until the adsorption reached equilibrium.

#### 2.5. Adsorption Kinetic and Isotherm Equations

#### 2.6. Cell Culture

_{2}, 37 °C constant temperature culture conditions. When the cells grew to cover 80–90% of the bottom of the flask, they were digested with trypsin and passaged every three days.

#### 2.7. Cytotoxicity Assays

#### 2.7.1. Cell Viability

_{e}and Ac represented the absorbance of the experimental group and the control group, A

_{0}and A

_{p}were the absorbances of the culture medium and solution of the PS-MPs dispersed in the medium, respectively.

#### 2.7.2. Cell Cycle

^{6}cells/mL, and then fixed with 70% cold ethanol. Cells were incubated with a propidium iodide staining working solution in the dark for 1 h. The red fluorescence of 10,000 events of propiodium iodide-stained cells was countered using a Cytoflex-S flow cytometer (Beckman Coulter, Inc. in Brea, CA, USA). The percentage of cells in the different phases of the cell cycle was calculated using Kaluza Analysis Software (Beckman Coulter, Inc. in Brea, USA).

#### 2.7.3. Apoptosis

#### 2.7.4. Mitochondrial Membrane Potential (MMP)

#### 2.7.5. Reactive Oxygen Species (ROS)

#### 2.8. Statistical Analysis

## 3. Results

#### 3.1. Characterization of PS-MPs

^{−1}and 750 cm

^{−1}was attributed to the C-H stretching vibration mode and the out-of-plane bending vibration mode on the benzene ring in the polystyrene molecule; The 2850 cm

^{−1}frequency belongs to the CH

_{2}symmetric stretching vibration mode in the polystyrene molecule, and 960 cm

^{−1}is attributed to the out-of-plane deformation vibration mode of the olefin in the polystyrene molecule; the prominent peak at around 1455 cm

^{−1}and 1730 cm

^{−1}was attributed to the vibration mode of the benzene ring in the polystyrene molecule. The composition and characteristic diffraction peaks of the PS-MPs corresponded to previous reports [22].

**Figure 1.**Characterization of PS−MPs used in this study. (

**A**) SEM images of PS−MPs with different particle sizes: 0.1 μm (

**a**); 1 μm (

**b**); 10 μm (

**c**); 50 μm (

**d**); 100 μm (

**e**). (

**B**) FTIR spectra of PS−MPs.

Particle Size (μm) | 0.1 | 1 | 10 | 50 | 100 |
---|---|---|---|---|---|

Surface area (m^{2}/g) | 62.248 | 38.193 | 16.887 | 3.824 | 3.351 |

Total pore volume (cc/g) | 0.336 | 0.476 | 0.043 | 0.007 | 0.004 |

Average pore diameter (nm) | 19.970 | 9.818 | 10.013 | 7.082 | 5.096 |

#### 3.2. Effect of Reaction Time on Adsorption and Desorption of NP on PS-MPs

#### 3.3. Intrinsic and Extrinsic Factors That Influence NP Adsorption to PS-MPs

^{+}increased the most.

**Figure 3.**The effect of PS-MP particle size (

**A**), initial NP concentration (

**B**), solution pH (

**C**), and metal ions contained in the solution (

**D**) on the adsorption process of NP. * indicates the significant difference between the treatment group (0.1 μm PS-MPs, initial NP concentration of 4 mg/L, pH 7 and control without metal ions, respectively) and other groups (* p < 0.05, ** p < 0.001, *** p < 0.0005).

#### 3.4. Adsorption Kinetics

^{2}) values. The intra-particle diffusion kinetic model was more suitable for 0.1 μm PS-MPs (R

^{2}= 0.988) than for other particle sizes of the PS-MPs. The data from the intra-particle diffusion rate constant k

_{1p}and the piece-wise fitting, C, of the 0.1 μm PS-MPs showed that the NP easily diffused inside the PS-MPs, whereas intra-particle diffusion was not the only rate-limiting step. Surprisingly, the Bangham kinetic model also gave good fittings for the adsorption of NP by 0.1 μm PS-MPs, indicating that intraparticle diffusion is crucial for NP adsorption by PS-MPs with small particle size. The kinetic model fitting curves mentioned here can be found in Appendix A Figure A1.

#### 3.5. Adsorption Isotherm

_{L}of the D-R isotherm indicated the adsorption behavior of the PS-MPs was extremely favorable for NP. The adsorption isotherm model-fitting curve mentioned here can be found in Appendix A Figure A2.

#### 3.6. Cell Proliferation and Apoptosis

#### 3.6.1. Cell Viability

#### 3.6.2. Cell Cycle

#### 3.6.3. Apoptosis

**Figure 5.**Effects of 48 h treatment of PS-MPs, NP and synergistics on the cell cycle and apoptosis of the Caco-2 cells. (

**A**) Shows the proportion of cells in the G1 phase via different exposures. (

**B**) Showed the sum of the cells in the S and G2 phases via different exposures. Early and late apoptotic Caco-2 cells were analyzed by flow cytometry after Annexin V and PI staining. A statistical graph of the percentage of cells in each phase was calculated (

**C**). * indicates the significant difference between the treatment group and the control group (** p < 0.001, *** p < 0.0005).

^{#}indicates the significant difference between the marked groups (

^{#}p < 0.05,

^{###}p < 0.0005).

#### 3.7. Cell Function

#### 3.7.1. Mitochondrial Depolarization

#### 3.7.2. Reactive Oxygen Species Production

## 4. Discussion

## 5. Conclusions

## Author Contributions

## Funding

## Data Availability Statement

## Conflicts of Interest

## Appendix A

Name | Equations | References |
---|---|---|

kinetics | ||

Pseudo-first-order | $\mathrm{ln}\left({\mathrm{q}}_{\mathrm{e}}-{\mathrm{q}}_{\mathrm{t}}\right)=\mathrm{ln}{\mathrm{q}}_{\mathrm{e}}-{\mathrm{k}}_{1}\mathrm{t}$ | [40] |

Pseudo-second-order | $\frac{\mathrm{t}}{{\mathrm{q}}_{\mathrm{t}}}=\frac{1}{{\mathrm{k}}_{2}{\mathrm{q}}_{\mathrm{e}}^{2}}+\frac{1}{\mathrm{q}}\mathrm{t}$ | [41] |

Intra-particle diffusion | ${\mathrm{q}}_{\mathrm{t}}={\mathrm{k}}_{\mathrm{ip}}{\mathrm{t}}^{0.5}+\mathrm{c}$ | [42] |

Bangham | ${\mathrm{q}}_{\mathrm{t}}={\mathrm{q}}_{\mathrm{e}}\left(1-{\mathrm{e}}^{-\mathrm{kt}}{}^{\mathrm{z}}\right)$ | [43] |

isotherm | ||

Langmuir | $\frac{{\mathrm{c}}_{\mathrm{e}}}{{\mathrm{q}}_{\mathrm{e}}}=\frac{1}{{\mathrm{k}}_{\mathrm{l}}{\mathrm{q}}_{\mathrm{m}}}+\frac{{\mathrm{c}}_{\mathrm{e}}}{{\mathrm{q}}_{\mathrm{max}}}$ | [44] |

Freundlich | $\mathrm{ln}{\mathrm{q}}_{\mathrm{e}}=\mathrm{ln}{\mathrm{k}}_{\mathrm{F}}+\frac{1}{{\mathrm{n}}_{\mathrm{f}}}\mathrm{ln}{\mathrm{c}}_{\mathrm{e}}$ | [45] |

D-R | $\mathrm{ln}{\mathrm{q}}_{\mathrm{e}}=\mathrm{ln}{\mathrm{q}}_{\mathrm{m}}-{\mathrm{K}}_{\mathrm{DR}}{\mathsf{\epsilon}}^{2}$ $\mathsf{\epsilon}=\mathrm{RT}\mathrm{ln}\left(1+\frac{1}{{\mathrm{C}}_{\mathrm{e}}}\right)$ $\mathrm{E}=\frac{1}{\sqrt{2{\mathrm{K}}_{\mathrm{DR}}}}$ | [46] |

_{e}and q

_{t}denote the adsorption capacity of the PS-MPs at the condition of equilibrium and at the time t (h); k

_{1}(h

^{−1}) is the rate invariable of the pseudo-first-order adsorption kinetic models; k

_{2}(g·mg

^{−1}·h

^{−1}) is the pseudo-second-order rate invariable; k

_{ip}(mg·g

^{−1}·h

^{−0.5}) represents the rate invariable of intra-particle diffusion; C is a constant related to thickness and boundary layer. K is the adsorption rate constant of the Bangham model, and Z is the adsorption constant. q

_{e}(mg·g

^{−1}) is the equilibrium adsorption capacity of NP per unit adsorbent; ce (mg·L

^{−1}) is the equilibrium concentration of NP insolution; k

_{L}(L·mg

^{−1}) is the Langmuir adsorption constant; q

_{max}(mg·g

^{−1}) represents the theoretical maximum adsorption capacity; n

_{f}is the surface heterogeneity factor and k

_{F}(mg·g

^{−1}) is the Freundlich partition co-efficient; K

_{DR}is the constant related to adsorption energy (mol

^{2}·KJ

^{−2}); ε is Polanyi potential energy (KJ·mol

^{−1}); R is the gas constant, T is the absolute temperature (K), and E is the average free energy of adsorption (KJ·mol

^{−1}), |E| < 8 (KJ·mol

^{−1}) is physical adsorption, |E| > 16 (KJ·mol

^{−1}) is chemisorption.

**Figure A1.**The plots of adsorption kinetics fitted by pseudo-second-order kinetic (

**A**), intra−particle diffusion kinetics (

**B**) and Bangham modes (

**C**). The adsorption of NP (4 mg/L) and PS−MPs (20 μg/mL) of different particle sizes were studied.

**Figure A2.**Adsorption isotherm model fitting of 20 μg/mL 0.1 μm of MPs−PS with different concentrations of NP. (

**A**) Langmuir model fitting. (

**B**) D−R model fitting.

**Figure A3.**The mitochondrial membrane potential depolarization of Caco−2 cells treated by 40 μmol/L NP, 500 mg/L PS-MPs with different particle sizes, 0.1 μm PS−MPs that absorbed NP at 48 h. Representative photograph from an inverted fluorescence microscope.

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**Figure 2.**Effects of reaction time on the adsorption and desorption of NP in PS−MPs. (

**A**) Adsorption process of NP solutions (4 mg/L) with 20 μg/mL PS−MPs with different particle size (0.1, 1, 10, 50, and 100 μm). (

**B**) Effect of different initial NP concentrations (4 and 20 mg/L) on time-sorption capacity trends. (

**C**) The desorption behavior of NP on the PS−MPs in the water environment (pH 7.0, 3.5% sodium chloride, 25 ± 2 °C and 150 r/min) and the warm-blood body gastrointestinal environment (pH = 2.8, 15.5 mmol/L sodium taurocholate, 10 g/L of pepsin, 35 ± 2 °C and 100 r/min) was investigated.

**Figure 4.**Effects of PS-MPs, NP, and synergistics on the cell viability of Caco-2 cells. Cells were incubated alone with different particle sizes of 500 mg/L PS-MPs (

**A**) and different concentrations of NP (

**B**) for 12 and 48 h. The effect of 0.1 μm PS-MPs alone and in combination with NP on Caco-2 cell viability at 12 h and 48 h (

**C**). Cell viability was measured by CCK-8 assay. * indicates the significant difference between the treatment group and the control group (* p < 0.05, ** p < 0.001, *** p < 0.0005).

^{#}indicates the significant difference between the marked groups (

^{#}p < 0.05,

^{###}p < 0.0005).

**Figure 6.**The mitochondrial membrane potential depolarization of Caco-2 cells treated with 40 μmol/L NP, 500 mg/L PS-MPs (with different particle sizes), and 0.1 μm PS-MPs that absorbed NP at 48 h; the fluorescence values were calculated by Image J software (National Institute of Mental Health, USA) and the ratio of red to green fluorescence reflected the degree of mitochondrial membrane depolarization (2). * indicates the significant difference between the treatment group and the control group (** p < 0.001, *** p < 0.0005).

^{#}indicates the significant difference between the marked groups (

^{###}p < 0.0005).

**Figure 7.**Effects of PS-MPs and NP on the generation of the intracellular ROS of Caco-2 cells at 48 (

**A**) and 72 h (

**B**), expressed as a ratio to the control group. * indicates the significant difference between the treatment group and the control group (* p < 0.05, *** p < 0.0005).

^{#}indicates the significant difference between the marked groups (

^{##}p < 0.001,

^{###}p < 0.0005).

**Table 2.**Kinetic parameters of NP adsorption by the PS-MPs obtained from the pseudo-second-order, Intra-particle diffusion and Bangham models.

Kinetic Model | Particle Size (μm) | |||||
---|---|---|---|---|---|---|

0.1 | 1 | 10 | 50 | 100 | ||

Pseudo-second-order model | ||||||

k_{2} (g·mg^{−1}·h^{−1}) | 127.567 | 27.779 | 18.979 | 66.961 | 13.285 | |

q_{e} (mg·g^{−1}) | 193.923 | 193.870 | 193.859 | 193.851 | 189.560 | |

R^{2} | 0.9998 | 0.9998 | 0.9997 | 0.9982 | 0.9987 | |

Intra-particle diffusion model | ||||||

k_{ip} (mg·g^{−1} min^{−1}) | k_{1p} | 2.10013 | 0.15685 | 0.26952 | 1.30454 | 0.15907 |

k_{2p} | 0.20992 | 0.04903 | −0.11312 | 0.02179 | −0.55142 | |

k_{3p} | −0.01188 | −0.09727 | −0.01576 | −0.12874 | 0.19729 | |

C (mg·g^{−1}) | C_{1} | 191.949 | 192.81716 | 192.65587 | 192.38299 | 188.71922 |

C_{2} | 193.385 | 193.36525 | 194.11567 | 193.68406 | 191.49437 | |

C_{3} | 193.944 | 194.37526 | 193.43371 | 194.81593 | 181.1686 | |

R^{2} | 0.9875 | 0.4645 | 0.8277 | 0.9451 | 0.2302 | |

Bangham model | ||||||

k | 6.35439 | 5.47685 | 5.40951 | 6.52911 | 7.89394 | |

z | 0.11868 | 0.02838 | 0.03225 | 0.12528 | 0.3395 | |

R^{2} | 0.9519 | 0.2615 | 0.5853 | 0.7581 | 0.4534 |

Isotherm Model | |||||||||
---|---|---|---|---|---|---|---|---|---|

Langmuir Model | |||||||||

q_{max} (mg·g^{−1}) | K_{L} | R^{2} | R_{L} | ||||||

1 | 4 | 10 | 20 | 30 | 40 | 50 | |||

1665.6118 | 3.9657 | 0.9880 | 0.2014 | 0.0593 | 0.0246 | 0.0125 | 0.0083 | 0.0067 | 0.0050 |

D-R model | |||||||||

q_{max} (mg·g^{−1}) | K_{D-R} (mol^{2}·KJ^{−2}) | R^{2} | E | ||||||

19.5600 | 1.52938 × 10^{8} | 0.8062 | 5.717 × 10^{−5} |

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## Share and Cite

**MDPI and ACS Style**

Ding, F.; Zhao, Q.; Wang, L.; Ma, J.; Song, L.; Huang, D.
Adsorption Behavior of Nonylphenol on Polystyrene Microplastics and Their Cytotoxicity in Human Caco-2 Cells. *Water* **2022**, *14*, 3288.
https://doi.org/10.3390/w14203288

**AMA Style**

Ding F, Zhao Q, Wang L, Ma J, Song L, Huang D.
Adsorption Behavior of Nonylphenol on Polystyrene Microplastics and Their Cytotoxicity in Human Caco-2 Cells. *Water*. 2022; 14(20):3288.
https://doi.org/10.3390/w14203288

**Chicago/Turabian Style**

Ding, Fangfang, Qianqian Zhao, Luchen Wang, Juan Ma, Lingmin Song, and Danfei Huang.
2022. "Adsorption Behavior of Nonylphenol on Polystyrene Microplastics and Their Cytotoxicity in Human Caco-2 Cells" *Water* 14, no. 20: 3288.
https://doi.org/10.3390/w14203288