Effects of Ti₃C₂T_x (MXene) on growth, oxidative stress, and metabolism of Microcystis aeruginosa

Abstract

The potential ecotoxicity of Ti₃C₂T_x (MXene) is becoming a growing concern due to its widespread use in the field of environmental remediation. Unfortunately, little is known about the toxic effects and mechanisms of Ti₃C₂T_x on aquatic phytoplankton. Herein, we investigated the influence of Ti₃C₂T_x on the growth, oxidative stress, and metabolism of the phytoplankton Microcystis aeruginosa using conventional toxicological and metabolomics methods. Results showed that Ti₃C₂T_x had a dose-dependent effect on the physiological ecology of M. aeruginosa. Although low Ti₃C₂T_x concentrations (≤1 mg L⁻¹) did not significantly change the M. aeruginosa growth, oxidative status, and cell morphology, high concentrations (≥5 mg L⁻¹) substantially reduced its proliferation and photosynthetic capacity. The metabolomics results showed that low (1 mg L⁻¹) and high (5 mg L⁻¹) Ti₃C₂T_x concentrations induced the expression of 43 and 128 differential metabolites in M. aeruginosa, respectively, which were mainly enriched in the amino acid metabolism and lipid metabolism pathways. These results suggest that Ti₃C₂T_x resulted in metabolic disorders in M. aeruginosa, such as porphyrin and chlorophyll metabolism and glycerophospholipid metabolism, thereby inhibiting the photosynthetic activity of M. aeruginosa and ultimately leading to a decrease in algal growth. This study provides new insights into the toxicity mechanism of Ti₃C₂T_x against M. aeruginosa, which helps us understand the potential risks of Ti₃C₂T_x in the aquatic environment.

---

Environmental significance

The widespread use of MXene nanomaterials resulted in their inevitable release into the environment, potentially threatening the safety of the aquatic environment and human health. There has been concern about the ecotoxicity of Ti₃C₂T_x (MXene), but little is known about the effects of Ti₃C₂T_x toxicity on phytoplankton. This study found that Ti₃C₂T_x can inhibit the growth and photosynthetic capacity of M. aeruginosa by disrupting porphyrin and chlorophyll metabolism, and defend against oxidative stress via amino acid metabolic pathways. These findings offer significant implications in better understanding the Ti₃C₂T_x-induced M. aeruginosa toxicity and its potential risks in the aquatic environment.

1. Introduction

MXenes are novel two-dimensional (2D) nanomaterials consisting of transition metal nitrides, carbides, and carbon nitrides.¹ M_N+1X_nT_X is the general formula for MXene (n = 1–3), where M denotes an early transition metal (e.g., Ti, Zr, and Cr), X represents carbon or nitrogen, and T signifies the surface group (e.g., –OH, =O, or –F).² Due to the high stability, flexibility, and ion-exchange properties of these materials, MXenes are used in numerous applications, including water purification,³ sensors,⁴ adsorbents,⁵ catalysis,⁶ thermoelectrics,⁷ capacitors,⁸ and biomedicine.^9,10 During the production, utilization, and disposal of MXenes, they could be released into the environment through wastewater discharge pathways or as by-products of various applications (e.g., photocatalysts and adsorbents).^11,12 The unique physicochemical properties of MXenes are likely to have an unpredictable ecological impact. Therefore, while recognizing the positive benefits of MXenes, their possible negative environmental impact is attracting increasing research attention.

Ti₃C₂T_x is one of the most used and researched MXenes. Studies have shown that 20 mg L⁻¹ Ti₃C₂T_x exhibits high adsorption efficiency for metal Pb,¹³ while 100 mg L⁻¹ Ti₃C₂T_x can effectively remove Cr(VI) and MO from water.¹⁴ Due to its excellent adsorption properties, Ti₃C₂T_x is extensively utilized for aquatic environmental remediation.^15–17 However, the extensive use of Ti₃C₂T_x for aquatic environmental remediation has raised concerns about its ecological safety and its impact on organism health. One study showed that a high concentration (200 mg L⁻¹) of Ti₃C₂T_x nanosheets could lead to zebrafish embryo death via surface adsorption.¹¹ In another study, under acute exposure conditions, 10 mg L⁻¹ Ti₃C₂T_x accumulated in the intestines of Daphnia magna, leading to metabolic disorders.¹⁸ Therefore, the effect of Ti₃C₂T_x on aquatic organisms cannot be ignored. Although existing studies show that Ti₃C₂T_x threatens the health of vertebrates and invertebrates in the water column, little is known about its impact on aquatic phytoplankton. Phytoplankton are abundant in aquatic ecosystems and play a vital role in maintaining their stability. Therefore, revealing the impact of Ti₃C₂T_x on aquatic phytoplankton is crucial to comprehensively and systematically assess aquatic ecological security and the environmental risks of Ti₃C₂T_x.

Microcystis aeruginosa (M. aeruginosa) is one of the most widely distributed microalgae.¹⁹ Due to its simple structure, short growth cycle, and easy cultivation and observation, M. aeruginosa has become an important indicator species for assessing the effect of nanomaterials on aquatic phytoplankton.²⁰ Studies have shown that exposure to nanomaterials (e.g., graphene oxide, nanoplastics, cerium oxide, titanium dioxide, and zinc oxide) induces a complex, diverse range of toxic effects on M. aeruginosa, including growth inhibition,²¹ oxidative damage,^22,23 and extracellular secretion from the algal cells.^24,25 Although extensive research has investigated the impact of nanomaterials on M. aeruginosa, minimal studies are available involving the effect of Ti₃C₂T_x nanomaterials.

Metabolomics is a science that studies the changes in metabolites when organisms are stimulated or disturbed, and is used to detect small molecules with a relative molecular mass of less than 1000 and clarifies the effect of the interactions between organisms and their surroundings at the molecular level due to its high sensitivity and precision.²⁶ Metabolomics is currently successfully used to assess the mechanism behind the toxicity of nanomaterials to algae.^20,27 Therefore, this study mainly aims to reveal the toxic effect of Ti₃C₂T_x on M. aeruginosa using metabolomics. In addition, the potential toxicity mechanism of Ti₃C₂T_x against M. aeruginosa is further elucidated by examining the growth characteristics, photosynthetic parameters, antioxidant enzyme activity, and cell morphology of the algal cells. The results of this study can provide new insights into the metabolite characteristics and toxicity mechanism of Ti₃C₂T_x against algae in the aquatic environment, which is of great significance for evaluating the ecological and environmental health risks of the application of Ti₃C₂T_x in water treatment and environmental remediation.

2. Materials and methods

2.1 Ti₃C₂T_x materials

The colloidal solution of Ti₃C₂T_x nanosheets with diameters of 100 nm was purchased from XFNANO Materials Technology Co. Ltd., Nanjing, China. The original Ti₃C₂T_x was stored in a refrigerator in the dark at 4 °C until further use. To ensure the reliability of Ti₃C₂T_x materials purchased, the sizes and shapes of the Ti₃C₂T_x nanosheets were characterized using transmission electron microscopy (TEM, JIM-2100, Japan). X-ray diffractograms (XRDs) of Ti₃C₂T_x were analyzed using a Bruker D8 Venture (Bruker AXS, Germany). The absorption spectrum of the Ti₃C₂T_x solution was determined using a UV spectrophotometer (Jenway-6850, UK). A Brookhaven high-sensitivity zeta potential and size analyzer (NanoBrook 90plus PALS, Brookhaven Instruments Corporation, USA) was used to determine the zeta potential and hydration size of Ti₃C₂T_x in the exposure medium.

2.2 Algal culture and reagents

M. aeruginosa (FACHB-905) was purchased from the China Freshwater Algae Bank (Wuhan, China), Institute of Aquatic Biology, Chinese Academy of Sciences. The algae were cultured in sterile 250 mL triangular flasks containing a COMBO medium at 25 °C, a 12 h : 12 h light–dark ratio, and a 2500 lux light intensity. The culture medium was prepared according to the method described by Kilham et al.²⁸ and was sterilized at high temperature before use. The triangular flasks were shaken manually three times daily to prevent algal precipitation.

2.3 Exposure experiments

M. aeruginosa in the exponential growth phase was transferred to 250 mL of COMBO medium (Table S1†) in a sterile environment at an algal cell concentration of 10⁶ cells per mL and was cultured at 25 °C, a 12 h : 12 h light–dark ratio, and a 2500 lux light intensity.²⁹ Based on the environmental concentration of metal nanomaterials,³⁰ the Ti₃C₂T_x material was individually added to the algal culture flasks to final concentrations of 0 mg L⁻¹, 0.25 mg L⁻¹, 0.5 mg L⁻¹, 1 mg L⁻¹, 5 mg L⁻¹, and 10 mg L⁻¹. The reason for not using equal concentration gradients was mainly due to the nonlinear interaction between the MXene and algal growth.⁵ Three biological replicates were set under each concentration treatment group. The experiment lasted for 168 h, after which the algal samples were collected to analyze the algal growth, photosynthetic parameters, morphology, antioxidant enzyme activity, and metabolomics.

2.4 Analysis of the algal cell growth and inhibition rate

A linear relationship was evident between the algal concentration and the optical density (OD) value,³¹ while M. aeruginosa displayed strong absorbance at 680 nm. Therefore, an OD of 680 nm (OD₆₈₀) was used as an indirect indicator of algal growth, which was estimated at a wavelength of 680 nm using a UV spectrophotometer (Shanghai Jinghua Technology Co., Ltd.). In order to obtain reliable data, a linear relationship between the algal density and the OD₆₈₀ value has been established in this study, as shown in Fig. S1.† The linear regression equation for OD values and algal density is y = 13.749x − 0.1353 (R² = 0.994), where x represents the OD₆₈₀ values and y represents the algal cell density. In addition, the inhibition of algal growth was calculated using a method described by Zhang et al.³² using eqn (1):where I% represents the algal growth inhibition rate. X_c and X_c0 denote the OD values of the control group at the end and beginning of exposure, respectively. X_t and X_t0 are the OD values of the exposed group at the end and beginning of exposure, respectively.

2.5 Measurement of the photosynthetic parameters and the morphological characterization of the algae

The photosynthetic activity of algal cells was measured using a Phyto-PAM fluorescence monitoring system (Walz, Germany). Briefly, 3 mL of the algal samples were stored in the dark for 5–10 min, after which the maximum light quantum yield (Fv/Fm) was measured. In addition, this study aimed to clarify whether the effect of Ti₃C₂T_x on the photosynthetic parameters of the algae was related to morphological algal cell damage. Algal cells were collected from the control group and several treatment groups after Ti₃C₂T_x exposure for 168 h. The morphology of the algal cells was photographed and analyzed using an optical microscope (BX53F2C, Olympus, Japan).

2.6 Analysis of the antioxidant activity

After exposure to Ti₃C₂T_x, the algal cells were collected via repeated freezing and thawing more than three times and centrifuged at 6000 rpm for 10 min at 4 °C. The collected sediment was mixed with PBS (0.01 M, pH 7.0), and the cells were homogenized via centrifugation. Finally, the supernatant was collected, and the malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and catalase (CAT) activity were determined using three different kits. The detailed operational steps for these indicators according to the instructions of the manufacturer (Biyuntian Biotechnology Co. Ltd., China) are provided in the ESI.†

2.7 Metabolomics analysis

The metabolomics analyses were performed according to a technique delineated by Wang et al.³³ The algal cells were collected after exposure to Ti₃C₂T_x for 168 h. The M. aeruginosa samples were transferred to centrifuge tubes, mixed with methanol (4 : 1 v/v), subjected to ultrasonic lysis, and centrifuged at 6000 rpm for 10 min at 4 °C. After collecting the supernatant, the remainder was washed with PBS and centrifuged again. This process was repeated three times, after which the microalgae were flash-frozen in liquid nitrogen, stored at −80 °C, and sent to Shanghai Meiji Biomedical Technology Co. Ltd. for untargeted metabolomics analysis. The extracted samples were allowed to stand for 30 min, after which they were centrifuged for 15 min (13 000g, 4 °C) to obtain the supernatant. Finally, an MS/MS (Vanquish Horizon system and Q 3Exactive HF-X, Thermo Scientific, USA) system was used to detect the sample set metabolites.

An ACQUITY UPLC HSS T3 (100 mm × 2.1 mm i.d., 1.8 μm; Waters, Milford, USA) column was employed for LC-MS. Mobile phase A consisted of 95% water + 5% acetonitrile (containing 0.1% formic acid), while mobile phase B comprised 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). Other parameters included a 0.40 mL min⁻¹ flow rate, a 10 μL injection volume, and a 45 °C column temperature. The mass spectrometry conditions included spray voltages of 5500 V and −4500 V in positive/negative modes and a scanning range of 50–1200 m/z. Separation was performed at a flow rate of 0.40 mL min⁻¹ with an injection volume of 10 μL.

2.8 Statistical analyses

The significant differences between the control and treatment groups were determined by assessing the M. aeruginosa growth, inhibition rates, and antioxidant activity indices via one-way analysis of variance (ANOVA) with post hoc multiple comparisons (Duncan's test) using GraphPad Prism 10.0.2 (USA). Values of P < 0.05 were considered statistically significant. The histological data were analyzed to determine the metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. VIP values > 1 and p-values < 0.05 were set as thresholds for differential metabolite selection.

3. Results

3.1 Characterization of Ti₃C₂T_x

The TEM results showed (Fig. S2a†) that Ti₃C₂T_x consisted of irregular flakes. The XRD spectral results showed (Fig. S2b†) that the Ti₃C₂T_x crystals displayed small particle sizes and high crystalline phase levels, and the intense peaks (002) of Ti₃C₂T_x were at 6.12° and 19.9°. The UV spectrophotometry results showed that the Ti₃C₂T_x solution had a maximum absorption peak at 265 nm (Fig. S2c†). After 7 d of exposure, the average hydrodynamic size of Ti₃C₂T_x was 0.69 ± 0.11 μm and the zeta-potential of Ti₃C₂T_x was −4.65 ± 3.41 mV in the exposure medium (Fig. S2d†).

3.2 The M. aeruginosa growth response and photosynthetic parameters

The growth results showed that Ti₃C₂T_x affected the M. aeruginosa proliferation in a dose-dependent manner (Fig. 1a and S3†). Although the M. aeruginosa growth decreased at a Ti₃C₂T_x concentration of ≤1 mg L⁻¹ compared to the control group, no statistically significant differences (P > 0.05) were evident. Ti₃C₂T_x treatment concentrations ≥5 mg L⁻¹ for 168 h rapidly decreased the M. aeruginosa growth to 0.42 and 0.20 from 0.79 in the control group, showing statistically significant differences (P < 0.05) (Table S2†). After exposure to a Ti₃C₂T_x concentration ≤1 mg L⁻¹ for 168 h, no significant changes were evident in the M. aeruginosa inhibition rate compared to the control group. However, Ti₃C₂T_x concentrations ≥5 mg L⁻¹ yielded significantly higher M. aeruginosa inhibition rates of 55.04% and 93.0% (Fig. 1b). Based on the percentage inhibition of algal growth, the calculated EC₅₀ value for Ti₃C₂T_x at 168 h was 2.85 mg L⁻¹.

Fig. 1 Effect of Ti₃C₂T_x on the growth of M. aeruginosa. (a) Changes in OD values of algal cells over time under different concentrations of Ti₃C₂T_x treatment. (b) The percentage inhibition of algal cell growth by Ti₃C₂T_x at different concentrations for 168 h. (c) The changes in photosynthetic efficiency of algal cells after treatment with different concentrations of Ti₃C₂T_x for 168 h. The different letters in Fig. 1b and c indicate significant differences (p < 0.05) between different treatment groups.

The photosynthetic parameter results showed that a higher Ti₃C₂T_x concentration reduced the photosynthetic efficiency of M. aeruginosa (Fig. 1c). Compared with the control group, no significant changes (P > 0.05) were evident in the Fv/Fm values of M. aeruginosa in the 0.25 mg L⁻¹, 0.5 mg L⁻¹, and 1 mg L⁻¹ Ti₃C₂T_x groups throughout the exposure period, while those in the 5 mg L⁻¹ and 10 mg L⁻¹ Ti₃C₂T_x groups decreased substantially (P < 0.05) after exposure for 168 h.

3.3 Observation of the M. aeruginosa cell morphology

No significant changes were evident in the M. aeruginosa cell morphology after Ti₃C₂T_x exposure (Fig. S4†). M. aeruginosa presented spherical, smooth, intact algal cells in the control group, with an average diameter of 4.81 μm. The M. aeruginosa cells displayed no morphological alterations after exposure to Ti₃C₂T_x concentrations of 1 mg L⁻¹ and 5 mg L⁻¹, while only minimal changes were evident at 10 mg L⁻¹.

3.4 Oxidative damage to M. aeruginosa

M. aeruginosa showed no significant oxidative damage after Ti₃C₂T_x exposure (Fig. 2). Compared to the control group, no significant changes were evident in the M. aeruginosa MDA content, CAT activity, and SOD activity at Ti₃C₂T_x concentrations of 1 mg L⁻¹, 5 mg L⁻¹, and 10 mg L⁻¹ (Fig. 2).

Fig. 2 The effect of different concentrations of Ti₃C₂T_x on antioxidant biomarkers of M. aeruginosa. (a) Changes in MDA content. (b) Changes in CAT enzyme activity. (c) Changes in SOD enzyme activity.

3.5 M. aeruginosa metabolomic response

The metabolomics results showed that Ti₃C₂T_x exposure significantly altered the M. aeruginosa metabolites. Orthogonal partial least squares-discriminant analysis (OPLS-DA) (Fig. 3a) indicated that the first component explained 45.4% of the variation, while the second component accounted for 18%, with a more pronounced degree of separation between the samples of each treatment group (e.g., control, 1 mg L⁻¹, and 5 mg L⁻¹ Ti₃C₂T_x). This indicated significant differences between the metabolites of the different treatment groups. The Venn diagram results showed that 1 mg L⁻¹ Ti₃C₂T_x induced the differential expression of 43 M. aeruginosa metabolites, of which 35 were upregulated, and 8 were downregulated (P < 0.05). A 5 mg L⁻¹ Ti₃C₂T_x concentration caused the differential expression of 128 M. aeruginosa metabolites, of which 99 were upregulated, and 29 were downregulated (P < 0.05) (Fig. 3b).

Fig. 3 Metabolome changes of M. aeruginosa under Ti₃C₂T_x treatments. (a) OPLS-DA scores of metabolites of M. aeruginosa in different treatment groups in positive ion mode. (b) The Venn diagram shows the overlap of differential metabolites between the 1 and 5 mg L⁻¹ treatment groups. (c) Cluster analysis of differential expression of metabolites between the control and treatment groups.

The thermogram results (Fig. 3c) showed that 1 mg L⁻¹ Ti₃C₂T_x exposure upregulated several M. aeruginosa metabolites, including PE(LTE4/18:3(9Z,12Z,15Z)), PC(PGD2/20:4(5Z,8Z,11Z,14Z)), microcystin-LR, laminine, PI(22:6(4Z,7Z,11E,13Z,15E,19Z)-2OH(10S,17)/22:3(10Z,13Z,16Z)), PI(6ket-PGFIalpha/18:0), ginsenoside Rd, deoxycorticosterone acetate, ergothioneine, PI(PGFIalpha/16:0), ziziphin, PGP(18:0/PGEI), and N-(N-L-gamma-glutamyl-L-cysteinyl)glycine monoethyl ester. Furthermore, 1 mg L⁻¹ Ti₃C₂T_x exposure downregulated several M. aeruginosa metabolites, including pheophorbide a, MG(0:0/16:0/0:0), ginsenoside C-K, PI(PGD1/22:2(13Z,16Z)), and 2-acetic acid. Exposure to 5 mg L⁻¹ Ti₃C₂T_x upregulated various M. aeruginosa metabolites, including pheophorbide a, ginsenoside C-K, soyasaponin I, PC(18:0/LTE4), PI(PGD1/22:2(13Z,16Z)), erythromycin A enol ether, 2-acetic acid, dexniguldipine, PI(20:3(5Z,8Z,11Z)/TXB2), L-leucine, tinocrisposide, PA(22:5(7Z,10Z,13Z,16Z,19Z)/PGE2), PI(22:4(10Z,13Z,16Z,19Z), PI(22:6(4Z,7Z,11E,13Z,15E,19Z), and microcystin-LR. Exposure to 5 mg L⁻¹ Ti₃C₂T_x downregulated several M. aeruginosa, including PG(16:0/0:0)[U], ergothioneine, PC(LTE4/15:0), PA(22:6(4Z,8Z,10Z,13Z,16Z,19Z)-OH(7)/10:0), oleamide, and ustiloxin D.

3.6 Enrichment pathways of the M. aeruginosa metabolites

The KEGG classification results indicated that 10 metabolic pathways were significantly affected by the Ti₃C₂T_x exposure (Fig. 4). A 1 mg L⁻¹ Ti₃C₂T_x concentration stimulated the differential M. aeruginosa metabolites mainly involved in amino acid metabolism, global and overview maps, and nucleotide metabolism, while exposure to 5 mg L⁻¹ Ti₃C₂T_x influenced the differential metabolites associated with amino acid metabolism, lipid metabolism, and secondary metabolite biosynthesis (Fig. 4). Combined exposure to 1 mg L⁻¹ and 5 mg L⁻¹ Ti₃C₂T_x affected the differential M. aeruginosa metabolites correlated with amino acid metabolism.

Fig. 4 KEGG classification analysis of differentially expressed metabolites in M. aeruginosa induced by Ti₃C₂T_x. The left column is the number of metabolites affected by 1 mg L⁻¹, and the right column is the number of metabolites affected by 5 mg L⁻¹.

KEGG analysis showed that Ti₃C₂T_x significantly enriched the pathways of the M. aeruginosa differential metabolites related to metabolic processes (Fig. 5). Exposure to 1 mg L⁻¹ Ti₃C₂T_x caused significant M. aeruginosa differential metabolite enrichment, mainly in pathways associated with nucleotide metabolism, cofactor biosynthesis, amino acid metabolism (e.g. glycine, serine, threonine, alanine, aspartate, glutamate, cysteine, methionine, pyrimidine, and phenylalanine), purine metabolism, and the citrate cycle (TCA cycle) (Fig. 5a). A 5 mg L⁻¹ Ti₃C₂T_x concentration primarily stimulated significant M. aeruginosa differential metabolite enrichment in glycerophospholipid metabolism, amino acid metabolism (e.g., arginine, proline, lysine, phenylalanine, glycine, serine, and threonine), porphyrin metabolism, and ascorbate and aldarate metabolism (Fig. 5b). Combined exposure to 1 mg L⁻¹ and 5 mg L⁻¹ Ti₃C₂T_x stimulated significant M. aeruginosa differential metabolite enrichment, mainly during amino acid metabolism (e.g., glycine, serine, threonine, and phenylalanine) and glycerophospholipid metabolism.

Fig. 5 Enrichment pathway analysis of differentially expressed metabolites in M. aeruginosa induced by Ti₃C₂T_x. (a) 1 mg L⁻¹ Ti₃C₂T_x. (b) 5 mg L⁻¹ Ti₃C₂T_x. The horizontal coordinate is the rich factor, and the ordinate is the specific enrichment pathway. The p-value represents the enrichment significance of the pathway, and a P-value less than 0.05 is considered as a significant enrichment item. The size of the bubbles in the figure represents the enriched quantity of metabolites in the pathway.

4. Discussion

Microalgae are important primary producers in aquatic environments and play a vital role in maintaining the structure of these ecosystems.^34,35 Therefore, the physiological characteristics of algae are highly significant. This study showed that Ti₃C₂T_x exposure inhibited M. aeruginosa growth and stimulated the differential expression of many M. aeruginosa metabolites, which interfered with the related amino acid and lipid metabolism. Similarly, exposure to other nanomaterials, such as titanium dioxide, also significantly restricted M. aeruginosa growth and affected its physiological metabolic processes.²⁵ Furthermore, graphene and silver nanomaterials inhibited M. aeruginosa proliferation and interfered with amino acid, carbohydrate, and lipid metabolism,^29,36 while copper carbon nanocomposites disrupted lipid metabolism.³⁷ These findings suggest that nanomaterial exposure can significantly alter M. aeruginosa proliferation and physiological metabolic processes. As far as is known, this study is the first to reveal the effect of Ti₃C₂T_x on the growth and physiological metabolism of M. aeruginosa.

Biological indicators such as MDA and CAT are commonly used to reflect the oxidative damage caused by nanomaterials to the organism. Since MDA accumulation exacerbates the damage to the organism and accelerates algal cell death,³⁸ it is often used as a marker to assess oxidative stress severity.³⁹ CAT is another important antioxidant enzyme that significantly catalyzes H₂O production from H₂O₂, consequently protecting algae from the damage caused by excessive ROS.⁴⁰ Various studies have shown that nanomaterial exposure significantly alters the MDA content and CAT activity, leading to oxidative damage.^41–43 This study found that Ti₃C₂T_x exposure did not significantly alter the MDA content and CAT activity, suggesting that Ti₃C₂T_x did not cause substantial oxidative damage. Ti₃C₂T_x exposure-induced changes in M. aeruginosa amino acid metabolism are likely an important response during its defense against oxidative damage. This result may be related to the amino acid metabolic changes in M. aeruginosa induced by Ti₃C₂T_x exposure. Amino acids are involved in regulating the synthesis of antioxidant components as common precursors for specific protein biosynthesis.⁴⁴ Phenylalanine metabolism is a shared precursor for many secondary metabolites that contribute to the resistance to the oxidative stress induced by environmental toxins,⁴⁵ while threonine is effective in scavenging ROS accumulation.⁴⁶ The present study indicated that Ti₃C₂T_x exposure upregulated several metabolites (e.g., 5′-methylthioadenosine, dihydro-3-coumaric acid, and 5-aminovaleric acid) during phenylalanine and threonine metabolism. This suggests that Ti₃C₂T_x likely reduces oxidative damage in M. aeruginosa by increasing phenylalanine and threonine metabolism.

Ti₃C₂T_x exposure likely inhibits M. aeruginosa proliferation by interfering with porphyrin metabolism, which plays a vital role in algal photosynthesis and growth via the chlorophyll synthesis process.⁴⁷ Normal porphyrin metabolism ensures the synthesis of chlorophyll in microalgae, while abnormal porphyrin metabolism restricts this process, interfering with the optimal photochemical efficiency of photosystem II in microalgae, consequently inhibiting microalgal growth.^48,49 This study found that Ti₃C₂T_x exposure stimulated the differential expression of several metabolites in the porphyrin metabolic pathway of M. aeruginosa while also significantly inhibiting its maximum PSII photochemical quantum yield and proliferation. This suggests that Ti₃C₂T_x reduces the maximum photochemical efficiency of photosystem II by disrupting porphyrin metabolism in M. aeruginosa, reducing chlorophyll synthesis and ultimately inhibiting its photosynthetic activity and growth. Similarly, silver nanoparticles affected algal photosynthesis and proliferation by interfering with chlorophyll biosynthesis and metabolic degradation in the cysts.²⁹ The impact of Ti₃C₂T_x on the photosynthetic activity and growth of M. aeruginosa may also be related to its adsorption. A study has shown that flake-shaped MXenes can adsorb onto the surface of algal cells, which could effectively hinder the photosynthetic activity of algal cells and reduce their growth.⁵ Another study also suggested that spherical TiO₂ NPs significantly reduced the photosynthetic activity and growth in M. aeruginosa by aggregating on the algal surface and blocking the light.²⁵

Glycerophospholipid metabolism represents a key pathway during lipid metabolism disruption in M. aeruginosa by Ti₃C₂T_x. Lipid metabolism is a vital physiological metabolic pathway in organisms, which mainly involves glycerophospholipid, sphingolipid, and glycerol ester metabolic pathways.^33,50 Studies have shown that nanomaterial exposure can lead to lipid metabolism disorders by interfering with one or more of these pathways.⁵¹ In this study, Ti₃C₂T_x exposure stimulated the differential expression of multiple PE, PC, and PG class metabolites, which were mainly enriched in the glycerophospholipid pathway. This result suggests that Ti₃C₂T_x interferes with M. aeruginosa lipid metabolism, mainly via the glycerophospholipid metabolic pathway. Similarly, Ti₃C₂T_x also restricted Daphnia magna lipid metabolism via the glycerophospholipid pathway.¹⁸ PE, PC, and PG represent the main phospholipid components in cell membranes, playing a vital role in their integrity and stability. Their differential expression suggests that Ti₃C₂T_x interferes with algal cell membrane integrity.

Ti₃C₂T_x promotes the production of microcystin-LR in M. aeruginosa, which may increase its aquatic ecological risk. Microcystin-LR is one of the important metabolites of M. aeruginosa.^52,53 Current research found that exposure to Ti₃C₂T_x can induce an increase in microcystin-LR content in M. aeruginosa. Similarly, exposure to other nanomaterials (e.g., TiO₂ nanoparticles and ZnO nanoparticles) also led to an increase in microcystin-LR content in M. aeruginosa.^24,54 Research has shown that the release of microcystin-LR into the environment can cause various adverse effects on aquatic organisms, such as liver toxicity, intestinal toxicity, and neurotoxicity.^55–57 This suggests that exposure to Ti₃C₂T_x may increase its aquatic ecological risk by promoting the release of microcystin-LR from M. aeruginosa.

5. Conclusion

This study evaluates the toxic effect of Ti₃C₂T_x on M. aeruginosa using conventional toxicological indicators and metabolomics. We found that high Ti₃C₂T_x concentrations can inhibit the growth and photosynthetic efficiency of M. aeruginosa by disrupting the porphyrin and chlorophyll metabolic pathways (Fig. 6). The glycerophospholipid pathway and the glycine, serine, and threonine pathways are important pathways through which Ti₃C₂T_x causes phospholipid metabolism and amino acid metabolism disorders in M. aeruginosa, respectively (Fig. 6). These findings provide a new perspective for a deeper understanding of the toxicity and mechanisms of Ti₃C₂T_x on algae, which is of great value for evaluating the ecological and health effects of Ti₃C₂T_x on aquatic organisms. Nevertheless, this study still has some limitations. This study found that Ti₃C₂T_x can promote the synthesis of microcystin-LR, but the specific mechanism still needs further clarification. Therefore, in the future, we will further explore the mechanism of Ti₃C₂T_x promoting the synthesis and release of algal toxins by designing detailed experimental plans.

Fig. 6 Schematic diagram of the physiological interference mechanism of Ti₃C₂T_x on M. aeruginosa. Exposure to Ti₃C₂T_x can interfere with the physiological metabolism of M. aeruginosa, leading to a decrease in algal cell growth. The main pathways through which Ti₃C₂T_x induces physiological metabolic disorders in algae are glycerophospholipid metabolism, porphyrin and chlorophyll metabolism, and amino acid metabolism pathways.

Data availability

Data will be made available on request.

Author contributions

Qianqian Xiang: writing – original draft, conceptualization, visualization. Zhihao Ju: writing – original draft, investigation, methodology, data curation. Renhong Zhu: investigation, methodology. Minmin Niu: investigation, software. Yuanyuan Lin: validation, methodology. Xuexiu Chang: conceptualization, funding acquisition, writing – review & editing, supervision.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was financially supported by the Basic Research Project of Yunnan Province (202401AU070014, 2019FA043), the Kunming University Talent Programs (YJL2219), the Yunnan Provincial Education Department Scientific Research Fund Project (2023J0826), the International Joint Innovation Team for Yunnan Plateau Lakes and Laurentian Great Lakes, and the Yunnan Collaborative Innovation Center for Plateau Lake Ecology and Environmental Health.

References

1. X. Qu, Y. Guo, C. Xie, S. Li, Z. Liu and B. Lei, Photoactivated MXene nanosheets for integrated bone–soft tissue therapy: effect and potential mechanism, ACS Nano, 2023, 17, 7229–7240.
2. M. Ghidiu, M. Naguib, C. Shi, O. Mashtalir, L. M. Pan, B. Zhang, J. Yang, Y. Gogotsi, S. J. L. Billinge and M. W. Barsoum, Synthesis and characterization of two-dimensional Nb₄C₃ (MXene), Chem. Commun., 2014, 50, 9517–9520.
3. P. Srimuk, J. Halim, J. Lee, Q. Tao, J. Rosen and V. Presser, Two-dimensional molybdenum carbide (MXene) with divacancy ordering for brackish and seawater desalination via cation and anion intercalation, ACS Sustainable Chem. Eng., 2018, 6, 3739–3747.
4. Y. Cai, J. Shen, G. Ge, Y. Zhang, W. Jin, W. Huang, J. Shao, J. Yang and X. Dong, Stretchable Ti₃C₂T_x MXene/Carbon nanotube composite based strain sensor with ultrahigh sensitivity and tunable sensing range, ACS Nano, 2017, 12, 56–62.
5. M. Jakubczak, D. Bury, M. A. K. Purbayanto, A. Wójcik, D. Moszczyńska, K. Prenger, M. Naguib and A. M. Jastrzębska, Understanding the mechanism of Nb-MXene bioremediation with green microalgae, Sci. Rep., 2022, 12, 14366.
6. K. J. Gordon, X. D. Zhou and L. Fei, Carbonitride MXenes: an innovative catalyst support for sustainable hydrogen production, Chem Catal., 2023, 3, 100664.
7. M. Khazaei, M. Arai, T. Sasaki, M. Estili and Y. Sakka, Two-dimensional molybdenum carbides: potential thermoelectric materials of the MXene family, Phys. Chem. Chem. Phys., 2014, 16, 7841–7849.
8. L. Li, D. Zhang, J. Deng, Y. Gou, J. Fang, H. Cui, C. Zhang and M. Cao, Application of MXene-based materials in hybrid capacitors, Sustainable Energy Fuels, 2021, 5, 3278–3291.
9. C. Dai, H. Lin, G. Xu, Z. Liu, R. Wu and Y. Chen, Biocompatible 2D titanium carbide (MXenes) composite nanosheets for pH-responsive MRI-guided tumor hyperthermia, Chem. Mater., 2017, 29, 8637–8652.
10. X. Yu, X. Cai, H. Cui, S. W. Lee, X. F. Yu and B. Liu, Fluorine-free preparation of titanium carbide MXene quantum dots with high near-infrared photothermal performances for cancer therapy, Nanoscale, 2017, 9, 17859–17864.
11. G. K. Nasrallah, M. Al-Asmakh, K. Rasool and K. A. Mahmoud, Ecotoxicological assessment of Ti₃C₂T_x(MXene) using a zebrafish embryo model, Environ. Sci.:Nano, 2018, 5, 1002–1011.
12. S. Yu, H. Tang, D. Zhang, S. Wang, M. Qiu, G. Song, D. Fu, B. Hu and X. Wang, MXenes as emerging nanomaterials in water purification and environmental remediation, Sci. Total Environ., 2022, 811, 169533.
13. B. M. Jun, N. Her, C. M. Park and Y. Yoon, Effective removal of Pb(ii) from synthetic wastewater using Ti₃C₂T_x MXene, Environ. Sci.:Water Res. Technol., 2020, 6, 173–180.
14. P. Karthikeyan, K. Ramkumar, K. Pandi, A. Fayyaz, S. Meenakshi and C. M. Park, Effective removal of Cr(VI) and methyl orange from the aqueous environment using two-dimensional (2D) Ti₃C₂T_x MXene nanosheets, Ceram. Int., 2021, 47, 3692–3698.
15. R. Imsong and D. Dhar Purkayastha, Dual-functional superhydrophilic/underwater superoleophobic 2D Ti₃C₂T_X MXene-PAN membrane for efficient oil-water separation and adsorption of organic dyes in wastewater, Sep. Purif. Technol., 2023, 306, 122636.
16. J. A. Kumar, P. Prakash, T. Krithiga, D. J. Amarnath, J. Premkumar, N. Rajamohan, Y. Vasseghian, P. Saravanan and M. Rajasimman, Methods of synthesis, characteristics, and environmental applications of MXene: a comprehensive review, Chemosphere, 2022, 286, 131607.
17. L. Chen, M. Wakeel, T. Ul Haq, N. S. Alharbi, C. Chen and X. Ren, Recent progress in environmental remediation, colloidal behavior and biological effects of MXene: a review, Environ. Sci.:Nano, 2022, 9, 3168–3205.
18. Q. Xiang, Z. Wang, J. Yan, M. Niu, W. Long, Z. Ju and X. Chang, Metabolomic analysis to understand the mechanism of Ti₃C₂T_x (MXene) toxicity in Daphnia magna, Aquat. Toxicol., 2024, 270, 106904.
19. M. J. Harke, M. M. Steffen, C. J. Gobler, T. G. Otten, S. W. Wilhelm, S. A. Wood and H. W. Paerl, A review of the global ecology, genomics, and biogeography of the toxic cyanobacterium, Microcystis spp, Harmful Algae, 2016, 54, 4–20.
20. W. Zhang, J. Liu, Q. Li, Y. Xiao, Y. Zhang, N. Lei and Q. Wang, Effects of combined exposure of PVC and PFOA on the physiology and biochemistry of Microcystis aeruginosa, Chemosphere, 2023, 338, 139476.
21. X. Zheng, L. Zhang, C. Jiang, J. Li, Y. Li, X. Liu, C. Li, Z. Wang, N. Zheng and Z. Fan, Acute effects of three surface-modified nanoplastics against Microcystis aeruginosa: Growth, microcystin production, and mechanisms, Sci. Total Environ., 2023, 855, 158906.
22. E. Cruces, A. C. Barrios, Y. P. Cahue, B. Januszewski, P. Sepulveda, V. Cubillos and F. Perreault, Toxicity mechanisms of graphene oxide and cadmium in Microcystis aeruginosa: evaluation of photosynthetic and oxidative responses, Aquat. Toxicol., 2023, 263, 106703.
23. D. Wu, J. Zhang, W. Du, Y. Yin and H. Guo, Toxicity mechanism of cerium oxide nanoparticles on cyanobacteria Microcystis aeruginosa and their ecological risks, Environ. Sci. Pollut. Res., 2022, 29, 34010–34018.
24. Y. Tang, H. Xin, S. Yang, M. Guo, T. Malkoske, D. Yin and S. Xia, Environmental risks of ZnO nanoparticle exposure on Microcystis aeruginosa: Toxic effects and environmental feedback, Aquat. Toxicol., 2018, 204, 19–26.
25. D. Wu, S. Yang, W. Du, Y. Yin, J. Zhang and H. Guo, Effects of titanium dioxide nanoparticles on Microcystis aeruginosa and microcystins production and release, J. Hazard. Mater., 2019, 377, 1–7.
26. L. J. Zhang, L. Qian, L. Y. Ding, L. Wang, M. H. Wong and H. C. Tao, Ecological and toxicological assessments of anthropogenic contaminants based on environmental metabolomics, Environ. Sci. Ecotechnology, 2021, 5, 100081.
27. L. Qian, S. Qi, F. Cao, J. Zhang, F. Zhao, C. Li and C. Wang, Toxic effects of boscalid on the growth, photosynthesis, antioxidant system and metabolism of Chlorella vulgaris, Environ. Pollut., 2018, 242, 171–181.
28. S. S. Kilham, D. A. Kreeger, S. G. Lynn, C. E. Goulden and L. Herrera, COMBO a defined freshwater culture medium for algae and zooplankton, Hydrobiologia, 1998, 377, 147–159.
29. J. L. Zhang, Z. P. Zhou, Y. Pei, Q. Q. Xiang, X. X. Chang, J. Ling, D. Shea and L. Q. Chen, Metabolic profiling of silver nanoparticle toxicity in Microcystis aeruginosa, Environ. Sci.:Nano, 2018, 5, 2519–2530.
30. A. Syafiuddin, S. Salmiati, T. Hadibarata, A. B. H. Kueh, M. R. Salim and M. A. A. Zaini, Silver nanoparticles in the water environment in Malaysia: inspection, characterization, removal, modeling, and future perspective, Sci. Rep., 2018, 8, 986.
31. B. Du, G. Liu, M. Ke, Z. Zhang, M. Zheng, T. Lu, L. Sun and H. Qian, Proteomic analysis of the hepatotoxicity of Microcystis aeruginosa in adult zebrafish (Danio rerio) and its potential mechanisms, Environ. Pollut., 2019, 254, 113019.
32. C. Zhang, X. Lin, P. Gao, X. Zhao, C. Ma, L. Wang, H. Sun, L. Sun and C. Liu, Combined effects of microplastics and excess boron on Microcystis aeruginosa, Sci. Total Environ., 2023, 891, 164298.
33. P. Wang, Q. Q. Li, J. Hui, Q. Q. Xiang, H. Yan and L. Q. Chen, Metabolomics reveals the mechanism of polyethylene microplastic toxicity to Daphnia magna, Chemosphere, 2022, 307, 135887.
34. Z. Guo, J. Li and Z. Zhang, Meta-analysis for systematic review of global micro/nano-plastics contamination versus various freshwater microalgae: toxicological effect patterns, taxon-specific response, and potential eco-risks, Water Res., 2024, 258, 121706.
35. W. Yang, P. Gao, G. Ma, J. Huang, Y. Wu, L. Wan, H. Ding and W. Zhang, Transcriptome analysis of the toxic mechanism of nanoplastics on growth, photosynthesis and oxidative stress of microalga Chlorella pyrenoidosa during chronic exposure, Environ. Pollut., 2021, 284, 117413.
36. J. Zhao, Y. Liang, L. Jin, H. Zhang, B. Shi and X. Tang, Joint toxic action and metabolic mechanisms of graphene nanomaterial mixtures in Microcystis aeruginosa, Pol. J. Environ. Stud., 2023, 32, 1447–1458.
37. P. Zong, Y. Liu, H. Chen, S. Miao, K. Lian, C. Li, H. Zhang and M. Zhang, Inhibitory mechanism of nano-copper carbon composite on Microcystis aeruginosa, Algal Res., 2022, 68, 102877.
38. Y. Lu, X. Jiang, H. Xu, C. Liu, Y. Song, K. Pan, L. Wang, L. Du and H. Liu, Effects of 4-tert-butylpyrocatechol and tea polyphenol on growth, physiology and antioxidant responses in Microcystis aeruginosa, Aquat. Toxicol., 2023, 260, 106541.
39. L. Yuan, C. J. Richardson, M. Ho, C. W. Willis, B. P. Colman and M. R. Wiesner, Stress responses of aquatic plants to silver nanoparticles, Environ. Sci. Technol., 2018, 52, 2558–2565.
40. X. Zheng, W. Zhang, Y. Yuan, Y. Li, X. Liu, X. Wang and Z. Fan, Growth inhibition, toxin production and oxidative stress caused by three microplastics in Microcystis aeruginosa, Ecotoxicol. Environ. Saf., 2021, 208, 111575.
41. A. Ale, J. M. Galdopórpora, M. C. Mora, F. R. de la Torre, M. F. Desimone and J. Cazenave, Mitigation of silver nanoparticle toxicity by humic acids in gills of Piaractus mesopotamicus fish, Environ. Sci. Pollut. Res., 2021, 28, 31659–31669.
42. W. El-Houseiny, M. F. Mansour, W. A. M. Mohamed, N. A. Al-Gabri, A. A. El-Sayed, D. E. Altohamy and R. E. Ibrahim, Silver nanoparticles mitigate Aeromonas hydrophila-induced immune suppression, oxidative stress, and apoptotic and genotoxic effects in Oreochromis niloticus, Aquaculture, 2021, 535, 736430.
43. M. M. Matouke and M. Mustapha, Bioaccumulation and physiological effects of copepods sp. (Eucyclop sp.) fed Chlorella ellipsoides exposed to titanium dioxide (TiO₂) nanoparticles and lead (Pb²⁺), Aquat. Toxicol., 2018, 198, 30–39.
44. V. Florencio-Ortiz, S. Sellés-Marchart, J. Zubcoff-Vallejo, G. Jander and J. L. Casas, Changes in the free amino acid composition of Capsicum annuum (pepper) leaves in response to Myzus persicae (green peach aphid) infestation. A comparison with water stress, PLoS One, 2018, 13, e0198093.
45. X. Jin, J. Pan, C. Zhang, X. Cao, C. Wang, L. Yue, X. Li, Y. Liu and Z. Wang, Toxic mechanism in Daphnia magna due to phthalic acid esters and CuO nanoparticles co-exposure: the insight of physiological, microbiomic and metabolomic profiles, Ecotoxicol. Environ. Saf., 2024, 277, 116338.
46. X. Li, T. Peng, L. Mu and X. Hu, Phytotoxicity induced by engineered nanomaterials as explored by metabolomics: perspectives and challenges, Ecotoxicol. Environ. Saf., 2019, 184, 109602.
47. S. Hörtensteiner, Chlorophyll breakdown in higher plants and algae.pdf, Cell. Mol. Life Sci., 1999, 56, 330–347.
48. R. Croce and H. van Amerongen, Light harvesting in oxygenic photosynthesis: structural biology meets spectroscopy, Science, 2020, 369, eaay2058.
49. L. X. Zhao, J. J. Hu, Z. X. Wang, M. L. Yin, Y. L. Zou, S. Gao, Y. Fu and F. Ye, Novel phenoxy-(trifluoromethyl)pyridine-2-pyrrolidinone-based inhibitors of protoporphyrinogen oxidase: design, synthesis, and herbicidal activity, Pestic. Biochem. Physiol., 2020, 170, 104684.
50. L. Shen, Q. Q. Li, Y. H. Kang, Q. Q. Xiang, X. Luo and L. Q. Chen, Metabolomics reveals size-dependent persistence and reversibility of silver nanoparticles toxicity in freshwater algae, Aquat. Toxicol., 2023, 258, 106471.
51. Q. Q. Xiang, H. Yan, X. W. Luo, Y. H. Kang, J. M. Hu and L. Q. Chen, Integration of transcriptomics and metabolomics reveals damage and recovery mechanisms of fish gills in response to nanosilver exposure, Aquat. Toxicol., 2021, 237, 105895.
52. R. Dai, Y. Zhou, Y. Chen, X. Zhang, Y. Yan and D. An, Effects of arginine on the growth and microcystin-LR production of Microcystis aeruginosa in culture, Sci. Total Environ., 2019, 651, 706–712.
53. X. Yu, C. Huang, L. Wu, S. Hua, J. Ye, L. Meng and C. Xu, Effects of the herbicides quizalofop-p-ethyl and quizalofop-ethyl on the physiology, oxidative damage, synthesis, and release of microcystin-LR in Microcystis aeruginosa, Sci. Total Environ., 2021, 776, 146036.
54. J. Zhang, L. Jiang, D. Wu, Y. Yin and H. Guo, Effects of environmental factors on the growth and microcystin production of Microcystis aeruginosa under TiO₂ nanoparticles stress, Sci. Total Environ., 2020, 734, 139443.
55. S. L. d. M. Calado, M. Vicentini, G. S. Santos, A. Pelanda, H. Santos, L. A. Coral, V. d. F. Magalhães, M. Mela, M. M. Cestari and H. C. Silva de Assis, Sublethal effects of microcystin-LR in the exposure and depuration time in a neotropical fish: Multibiomarker approach, Ecotoxicol. Environ. Saf., 2019, 183, 109527.
56. Y. Duan, D. Xiong, Y. Wang, H. Dong, J. Huang and J. Zhang, Effects of Microcystis aeruginosa and microcystin-LR on intestinal histology, immune response, and microbial community in Litopenaeus vannamei, Environ. Pollut., 2020, 265, 114774.
57. N. Luan, J. Zuo, Q. Niu, W. Yan, T.-C. Hung, H. Liu, Q. Wu, G. Wang, P. Deng, X. Ma, J. Qin and G. Li, Probiotic Lactobacillus rhamnosus alleviates the neurotoxicity of microcystin-LR in zebrafish (Danio rerio) through the gut-brain axis, Sci. Total Environ., 2024, 908, 168058.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4en01074d

‡ Qianqian Xiang and Zhihao Ju contributed equally to this paper.

---