Violet phosphorus nanosheets exhibit higher toxicity in the freshwater microalgae Tetradesmus obliquus than black phosphorus nanosheets

Abstract

The potential environmental risks of two-dimensional (2D) phosphorene nanomaterials are gaining attention as their promising applications continue to expand. Violet phosphorus (VP) has been demonstrated to be a more stable phosphorene nanomaterial compared to black phosphorus (BP). However, current research has primarily focused on the toxic effects of BP, with limited information available regarding the toxicity of VP. This study comparatively analyzed the ecotoxicity and mechanisms of action of environmentally relevant concentration exposures of the common green algae Tetradesmus obliquus to BP and VP nanosheets. The results revealed that VP exhibited a greater growth inhibitory effect on the algae compared to BP, which was linked to disruptions in cell membrane function. Both BP and VP induced intracellular oxidative stress responses, yet they did not cause oxidative damage to algal cells. Transcriptional responses suggested that the number of differentially expressed genes in the algae exposed to VP was 29 times higher than that in the algae exposed to BP. Metabolomic analysis indicated that the number of differentially expressed metabolites induced by VP exposure in the algae was twice as high as the changes induced by BP. Furthermore, integrated transcriptome and metabolome analyses highlighted significant differences between BP and VP in core pathways, key metabolites, and driving genes. The findings of this study underscore the importance of considering the impact of different types of phosphorene materials when assessing their environmental risks.

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Environmental significance

The rapid development of phosphorene nanomaterials such as black phosphorus (BP) and violet phosphorus (VP), presents significant technological potential. Their environmental safety remains however a critical concern. The unique physicochemical properties of these materials may lead to distinct environmental behaviors and toxicological outcomes, necessitating tailored risk assessments and regulatory frameworks. While BP has been closely monitored and studied, the toxicological effects of VP, a more stable and potentially widely used variant, are largely unexplored. Comparing the effects of BP and VP on organisms highlights the importance of addressing potential hazards to ensure sustainable development. Our findings confirmed at the phenotypic, cellular, and molecular levels that VP exhibited greater toxicity toward freshwater microalgae than BP. Such insights are essential for balancing technological innovation with environmental health, thereby guiding the safer applications of emerging nanomaterials.

Introduction

Black phosphorus (BP) has recently attracted considerable interest because of its unique physical characteristics, e.g., thickness-dependent bandgap, high mobility, highly anisotropic charge transport, and excellent biodegradability and biocompatibility.¹ These intrinsic characteristics have spurred extensive research in various fields, including energy storage and conversion, environmental remediation, and biomedicine.² Meanwhile, violet phosphorus (VP), a member of the phosphorus family,³ offers an intriguing alternative because of its high carrier mobility, anisotropy, wide bandgap, stability, and ease of exfoliation.⁴ Specifically, VP has been demonstrated to be a more stable two-dimensional (2D) nanomaterial compared to BP,⁵ offering immense potential for applications in electronics, optoelectronics, and biomedicine.^6,7 Currently, research on VP is in its early stages,⁸ and there is a promising outlook for it to surpass BP as the new “wonder material”. However, as these novel nanomaterials find broader applications,⁹ concerns regarding their environmental hazards, health risks, and toxicity mechanisms have come under scrutiny.^10–13

The aquatic environment represents a significant sink for novel nanomaterials.¹⁴ For instance, these nanomaterials can enter water bodies through industrial emissions or via discharged wastewater.¹⁵ The rising production volume of phosphorus nanomaterials will increase the releases into aquatic environments, potentially harming human health and wildlife.^16,17 It is important to note that, to the best of our knowledge, measured environmental concentrations of both BP and VP are not yet available in the scientific literature. This lack of monitoring data currently limits the ability to conduct a comprehensive environmental risk assessment of phosphorus nanomaterials. Despite environmental monitoring data for BP and VP currently being scarce, it is advisable, in accordance with the precautionary principle, to initiate their ecotoxicological investigations. A comparison of the toxicity of VP and BP is important, given the potential of VP to exceed BP in performance and serve as an alternative. In this context, it is essential to demonstrate that the potential benefits of VP are accompanied by possible adverse effects that are comparable to or lower than those of BP.

The release of phosphorus nanomaterials into aquatic ecosystems and their subsequent toxic effects on aquatic organisms are becoming major concerns.¹⁷ Aquatic organisms act as both sensitive indicators of water pollution and play a crucial role in maintaining the healthy functioning of aquatic ecosystems.^18,19 Previous studies have confirmed the toxic effects of BP on aquatic organisms. For instance, high concentrations (5 and 10 mg L⁻¹) of BP nanosheets inhibited the growth of the freshwater alga Chlorella vulgaris.²⁰ Additionally, BP nanosheets at 15, 45, and 75 mg L⁻¹ were found to accelerate apoptosis in C. vulgaris.²¹ Few-layered BP at concentrations of 2 and 20 mg L⁻¹ can adsorb onto the surface of the chorion and subsequently penetrate the zebrafish embryo, exerting effects at the genetic level.¹⁷ Compared to BP, there is a significant lack of studies on the potential toxicity of VP to aquatic organisms. A recent antimicrobial study has revealed that VP nanosheets exhibit superior antibacterial activity against pathogens including Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Escherichia coli pUC19 via cell membrane penetration combined with oxidative stress.²² Moreover, VP nanosheets have been shown to exhibit higher antibacterial activity compared to BP nanosheets, which can be attributed to their more distinctive structural properties.²² Hence, it is essential to determine whether VP poses a greater toxicological risk to other species in aquatic ecosystems than BP. It is scientifically essential to systematically compare and elucidate the differential toxic effects and mechanisms of BP versus VP at the organismal, cellular, and molecular levels in aquatic organisms.

In this study, our primary objective was to carry out a comparative analysis of the toxicity and mechanisms of few-layered BP and VP nanosheets (termed as BP and VP) at environmentally relevant concentrations on aquatic organisms. To achieve this goal, we selected Tetradesmus obliquus, a common and sensitive freshwater alga,²³ as our model aquatic organism for investigation. Although environmental benchmark data for phosphorus nanomaterials including BP and VP are currently lacking, we selected environmentally relevant concentrations of 0.01, 0.1, and 1 mg L⁻¹ for exposure studies based in analogy to other two-dimensional nanomaterials.^24,25 First, we characterized the physicochemical properties of BP and VP in the exposure medium. Subsequently, we examined the effects of BP and VP on the biomass and pigment content of algal cells through growth inhibition toxicity testing. Furthermore, we employed infrared spectroscopy to analyze the interactions of algal cells with BP and VP. Additionally, we utilized biochemical markers to elucidate the mechanisms underlying BP- and VP-induced membrane toxicity and the generation of oxidative stress responses within the algal cells. Finally, through the combined application of transcriptomics and metabolomics, we revealed the molecular responses triggered by toxicity of BP and VP on the algae. To the best of our knowledge, this research is the first to investigate the toxicity and underlying mechanisms of VP in aquatic species. Furthermore, this research could serve as a basis for enhancing our knowledge of the possible side effects linked to exposure to phosphorus nanomaterials. It is also important to perform a comparative analysis of BP and VP to assess their toxicological effects and tackle environmental safety issues related to the interactions between phosphorus nanomaterials and living organisms.

Materials and methods

Test materials and test media

BP (thickness: 1–10 layers, lateral size: 100 nm–5 μm) and VP (thickness: 1–10 layers, lateral size: 0.05–1 μm) dispersions in water were obtained from Nanjing Xianfeng Nanomaterials Science and Technology Co., Ltd. These dispersions were then stirred using a magnetic stirrer for a minimum of 1 h to ensure uniformity. Subsequently, the suspensions were stored at 4 °C for long-term preservation. The morphology and stability of the BP or VP suspensions in the testing medium were characterized. The Supplementary Information (SI) provides the detailed experimental methods used for the physicochemical analysis of BP and VP.

Growth inhibition bioassay

The unicellular freshwater alga, T. obliquus, was obtained from the Chinese Academy of Sciences Institute of Hydrobiology in Wuhan, China. The algal medium, maintained at a pH of 7.8 ± 0.2 and used as the test medium, was freshly prepared in accordance with OECD Technical Guideline 201 medium.²⁶ The exposure concentrations for the test materials were 0, 0.01, 0.1, and 1 mg L⁻¹. This concentration range was intentionally selected to simulate environmentally relevant levels of 2D nanomaterials.²⁴ Exponentially growing algae cells (with a final density of 3 × 10⁵ cells per mL) were added to control and treated suspensions. Control experiments consisting of single test materials were required to eliminate their absorbance effects. All flasks containing various test materials were incubated in an artificial growth chamber consistently at a temperature of 24 ± 1 °C for 96 h with a photoperiod of 12 h light (3000–4000 lx) and 12 h dark. Growth inhibition (%) was calculated by (1-the specific growth rate in the treatment/the mean specific growth rate in the control) × 100%. The contents of photosynthetic pigments, namely chlorophyll a, chlorophyll b, and carotenoids, were quantified, with the detailed experimental procedures provided in the SI.

Biomarker assays

Algal cells were harvested after 96 h of culture by centrifugation at 15 000 rpm for 10 minutes at 4 °C using a D3024 high-speed microcentrifuge (Scilogex, USA). The cells were then washed three times with fresh culture medium before performing three functional assays: cellular membrane permeability (CMP), total reactive oxygen species (ROS), and mitochondrial membrane potential (MMP). For the CMP assessment, algal cells were incubated with 10 μM fluorescein diacetate (FDA, Aladdin) for 30 min at 25 °C in the dark. Total ROS were detected using 10 μM DCFH-DA (Macklin Biochemical, China) under the same incubation conditions. MMP was measured following 30 min incubation with 10 μM rhodamine 123 (Rh 123, Aladdin) under the identical conditions. After each treatment, cells were washed and fluorescence intensity was measured using an F96PRO spectrophotometer (Shanghai Kingdak, China) at 485/530 nm (excitation/emission), with all results expressed as percentages relative to control fluorescence. Each test concentration was replicated three times.

For the antioxidative activity assays, test algae were harvested from a 10 mL culture after 96 h and centrifuged at 4000 rpm for 40 min. The precipitate was resuspended in 1 mL phosphate buffer (at pH 7.2, 0.05 mol L⁻¹) and sonicated to rupture the cells by a ultrasonic cell crusher (XO-650D, Xianou Tech, China; 650 W, 3 s pulse, and 3 s interval) in an ice bath for 10 min. The cell debris was then removed by centrifuging the mixture at 15 000 rpm for 10 min at 4 °C using the high speed D3024 micro-centrifuge, and biochemical assays were performed on the supernatant. Superoxide dismutase (SOD) and malondialdehyde (MDA) assays were performed using appropriate commercial kits purchased from the Nanjing Institute of Jiancheng Biological Engineering (Nanjing, China). Each test concentration was replicated three times.

Characterization of interactions of particles with algae

Fourier Transform Infra-Red (FTIR) analysis was performed to determine the change of biomacromolecules (e.g., lipids, proteins, and polysaccharides) on the surfaces of algal cells after treating with BP and VP. To reveal a more distinct effect, 1 mg L⁻¹ of the test material was selected for FTIR characterization. T. obliquus cells after 96 h of exposure to BP or VP were used for testing. The dried samples of algal cells were characterized by FTIR spectroscopy (Thermo Scientific, Nicolet iS10, USA).

Transcriptome analysis

To identify differentially expressed genes (DEGs) in T. obliquus following exposure to various treatments, mRNA was extracted from the control group (0 mg L⁻¹ BP or VP) and treatment groups (1 mg L⁻¹ BP and 1 mg L⁻¹ VP) after 96 h. Libraries were constructed using three qualified mRNA samples from both the control and treatment groups, and transcriptome sequencing was conducted using the Illumina platform (illumina nova seq X Plus). The ABI 7500 qRT-PCR (Applied Biosystems, USA) was employed to validate the results of the transcriptome sequencing, ensuring higher abundance and specificity of gene expression. The sequences of primers for the selected DEGs are listed in Table S1, with the GAPDH gene serving as the internal control.

Metabolome analysis

Non-targeted metabolomics was employed to investigate changes in intracellular metabolites within T. obliquus cells before and after exposure to 1 mg L⁻¹ BP and 1 mg L⁻¹ VP for 96 h. Following metabolite extraction, quantitative analysis was conducted using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system (ThermoFisher, Germany) coupled with either an Orbitrap Q Exactive™ HF mass spectrometer or an Orbitrap Q Exactive™ HF-X mass spectrometer (Thermo Fisher, Germany). The raw data files generated by UHPLC-MS/MS were processed using the Compound Discoverer 3.3 (CD3.3, ThermoFisher) to perform peak alignment, peak picking, and quantification for each metabolite.

Statistical analysis

All data are expressed as mean ± standard deviation (SD). A two-way analysis of variance (ANOVA) was conducted using IBM SPSS software (version 23.0, IBM Corp., Armonk, NY, USA) to evaluate the effects of nanomaterial type and observation time on the physicochemical characteristics of two phosphorus nanomaterials. A one-way ANOVA, followed by Dunnett's post hoc test, was applied to identify differences in toxicity endpoints between the control and treatment groups. Additionally, a two-way ANOVA with Dunnett's post hoc test was performed to assess the effects of nanomaterial type and exposure concentration on the toxicity induced by the two phosphorus nanomaterials.

Results and discussion

Physicochemical properties of BP and VP in the test medium

BP and VP exhibited distinct characteristics of lamellar and layered structures, as shown in Fig. 1A and B, respectively. Additionally, the edges of the nanosheets displayed exceptionally sharp features. Given the sharp edges of BP and VP nanosheets, along with the similar structural features observed in other 2D nanomaterials such as graphene,²⁷ it is plausible that these ultrathin, rigid nanostructures may cause physical damage to cell membranes through mechanical cutting or piercing effects.

Fig. 1 Transmission electron microscope images of suspensions of primary BP (A) and VP (B) in the test medium, with a scale bar of 0.2 μm; zeta potential (ζP) (C) and hydrodynamic diameter (HD) (D) of BP and VP suspensions in the test medium over time. All values in Fig. 1C and D are expressed as mean ± standard deviation (n = 6). The asterisk represents the difference of ζP or HD between the different time points within the same phosphorene nanomaterial (***p < 0.001). The number sign represents the difference of ζP or HD between BP and VP at the same time point (#p < 0.05 and ###p < 0.001).

The dispersion and stability of nanomaterials in water are crucial for their toxicological effects.²⁸ Zeta potential (ζP) and hydrodynamic diameter (HD) are important physicochemical parameters that influence the stability of dispersion and the agglomeration behavior of 2D nanomaterials.^29,30 BP and VP exhibited a negative charge, as shown in Fig. 1C. Over 96 h, the absolute value of ζP of the suspended particles experienced a significant decrease. Furthermore, the absolute ζP value of VP at both 0 h and 96 h was significantly greater than that of BP, implying that VP exhibited higher suspension stability in the test medium compared to BP. This was further supported by the HD analysis (Fig. 1D), which indicated that the initial HD value of VP was significantly lower than that of BP. Over 96 h, no significant change was observed in the HD value of VP. However, the HD value of BP decreased significantly during this time frame, suggesting that the BP particles likely experienced agglomeration in the water column. Transmission electron microscope analysis further revealed that BP exhibited a greater degree of agglomeration in the test medium compared to VP (Fig. S1). In addition, as determined from X-ray photoelectron spectroscopy (XPS) (Fig. S2 and Table S2), the BP and VP surfaces consisted of 27.6% P/72.4% O and 51.4% P/48.6% O, respectively. It was estimated by the XPS analysis that BP particles might undergo more severe degradation compared to VP particles, which might also have led to the disintegration of BP particle agglomerates.

Algal growth and photosynthetic inhibition by BP and VP

As illustrated in Fig. 2A, when T. obliquus cells were exposed to 0.1 and 1 mg L⁻¹ of VP, their cell density decreased significantly compared to the control group. Moreover, increasing the concentration of VP particles decreased the algal cell density. In contrast, no significant changes were observed in algal cell density following exposure to varying concentrations of BP. When exposed to 1 mg L⁻¹ of VP, the algae exhibited a significantly lower cell density compared to exposure to 1 mg L⁻¹ of BP. Li et al.²⁰ found that BP at 1 mg L⁻¹ promoted the growth of C. vulgaris over a period of 120 h. As shown in Fig. 2B, increasing the concentration of particles enhanced the growth inhibition effect of VP on the algae. At the particle concentration of 1 mg L⁻¹, the growth inhibition rate of algae resulting from exposed to VP was significantly higher than that caused by BP.

Fig. 2 Cellular density (A), growth inhibition rate (B), relative levels of chlorophyll a (C), chlorophyll b (D), carotenoids (E), and chlorophyll a + b + carotenoids (F) of T. obliquus exposed to the suspensions of BP and VP at the particle concentrations of 0.01, 0.1, and 1 mg L⁻¹. All values are expressed as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 for the treatment groups compared to the control. #p < 0.05, ##p < 0.01, and ###p < 0.001 for the BP exposure groups compared to the VP exposure groups at the same concentration. †p < 0.05 and ††p < 0.01 for comparison among the different concentrations in the same phosphorene nanomaterial exposure.

Further analysis revealed a decreasing trend in the contents of chlorophyll a, chlorophyll b, carotenoids, and the total of chlorophyll a + b + carotenoids within these algae as the concentration of VP increased (Fig. 2C–F). Specifically, algae exposed to 1 mg L⁻¹ of VP exhibited significantly reduced levels of chlorophyll a, carotenoids, and chlorophyll a + b + carotenoids compared to the control. However, exposing the algae to varying concentrations of BP did not cause a significant change in the contents of their photosynthetic pigments. Additionally, compared to exposure to 1 mg L⁻¹ BP, the algae exhibited a significantly lower content of photosynthetic pigments when exposed to 1 mg L⁻¹ VP.

Based on the analysis of the results obtained, it can be concluded that the VP exhibited greater growth and photosynthetic inhibitory effects on T. obliquus compared to the BP. A study by Shen et al.²² also addressed that VP nanosheets had higher antibacterial activity against E. coli and S. aureus than BP nanosheets.

Interactions of BP and VP with the algal cells

The superficial structures of T. obliquus cells exposed to 1 mg L⁻¹ of BP and VP were characterized (Fig. 3A–F). Compared to the controls, neither oxidation nor dissolution of the cells was observed, and no significant structural changes were detected in either the BP or VP groups. In contrast, after treatment with BP or VP, the FTIR spectra of the algal cells exhibited changes (Fig. 3G). This indicates that BP and VP may interact with algal cells through extracellular contact, potentially disrupting the biomacromolecules on the cellular surface. As shown in Fig. 3G, both the control and treatment groups displayed absorption peaks associated with lipids (C=O, 1742 cm⁻¹), proteins (amide I 1650 cm⁻¹, amide II 1540 cm⁻¹, and amide III 1240 cm⁻¹), as well as a peak related to polysaccharides (1045 cm⁻¹). Under VP exposure, the transmittance corresponding to the peaks associated with lipids and proteins was 0.1 to 0.6% higher than the transmittance of the control group and 0.8 to 2.2% higher than the transmittance of the BP exposure group, respectively. Notably, the peaks associated with lipids and proteins were slightly diminished in the VP exposure group, compared to the control and BP exposure groups. This suggests a likely minor damage to the cell membrane of T. obliquus when exposed to VP. The possible reason was that VP features numerous sub-nanoneedles along the flake periphery, whose high surface energy promotes interaction with the cell membranes of microorganisms.²²

Fig. 3 Superficial structures of T. obliquus cells exposed to control group (A and D), 1 mg L⁻¹ BP (B and E), and 1 mg L⁻¹ VP (C and F). FTIR spectrums of T. obliquus cells exposed to BP and VP with the particle concentration of 1 mg L⁻¹ (G) and cellular membrane permeability (CMP) in the algae exposed to the suspensions of BP and VP at the particle concentrations of 0.01, 0.1, and 1 mg L⁻¹ (H). All values in Fig. 3H are expressed as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 for the treatment groups compared to the control. ##p < 0.01 and ###p < 0.001 for the BP exposure groups compared to the VP exposure groups at the same concentration. †p < 0.05, ††p < 0.01, and †††p < 0.001 for comparison among the different concentrations in the same phosphorene nanomaterial exposure.

As illustrated in Fig. 3H, BP significantly induced an increase in the CMP at the studied concentrations. 1 mg L⁻¹ of VP significantly elevated the CMP, whereas 0.1 mg L⁻¹ of VP significantly reduced the CMP. In addition, CMP levels in the algal cells exposed to 0.01 and 0.1 mg L⁻¹ BP were significantly higher than those in the cells exposed to equivalent concentrations of VP. However, CMP levels were significantly lower in the cells exposed to 1 mg L⁻¹ BP compared to those exposed to 1 mg L⁻¹ VP. The layered structure of 2D nanomaterials has the potential to directly disrupt and impair cell membranes, ultimately leading to the leakage of intracellular contents and subsequent cell death.³¹ Generally, both BP and VP resulted in an increase in CMP, suggesting that the phosphorus nanomaterials may directly enter the cells.

Oxidative stress response induced by BP and VP

As illustrated in Fig. 4A, compared to the control group, BP significantly elevated the levels of ROS within the algal cells at the studied concentrations. The results of Dong et al.³² demonstrated a significant increase in intracellular ROS levels after 24 h of exposure to BP nanosheets in human cells across all studied concentrations (0.31–80 μg mL⁻¹). Additionally, the levels of ROS induced by BP at 0.01 and 1 mg L⁻¹ were significantly higher than those induced by VP. As shown in Fig. 4B, with increasing BP or VP concentrations, MMP gradually increased. This means that mitochondria were the main sites of cellular ROS production. Notably, the levels of MMP induced by both BP and VP at 0.1 and 1 mg L⁻¹ were significantly elevated compared to those in the control group. Furthermore, the MMP level induced by BP at 1 mg L⁻¹ was markedly higher than the MMP level induced by VP at the same concentration. Generally, the intracellular ROS induced by the phosphorus nanomaterials did not lead to mitochondrial destruction, suggesting that the amount of ROS they generated within cells was limited. Fig. 4C demonstrates that 1 mg L⁻¹ of VP significantly enhanced SOD activity. Moreover, the SOD activity in the algal cells exposed to 0.01 and 1 mg L⁻¹ VP was significantly higher than that in the cells exposed to the same concentrations of BP. These findings suggested that exposure to the VP activated the intracellular antioxidant defense system. Meanwhile, Fig. 4D indicates that there was no statistically significant change in MDA content observed in the algae exposed to BP and VP compared to the control group. This means that both BP and VP did not cause significant oxidative damage to the algal cells in the present study. Thus, it is reasonable to believe the antioxidant defense system was sufficiently robust to counteract the oxidative stress caused by ROS induced by the phosphorus nanomaterials.

Fig. 4 Relative levels of reactive oxygen species (ROS) (A), levels of mitochondrial membrane potential (MMP) (B), activities of superoxide dismutase (SOD) (C), and contents of malondialdehyde (MDA) (D) in T. obliquus exposed to the suspensions of BP and VP at the particle concentrations of 0.01, 0.1, and 1 mg L⁻¹. All values are expressed as mean ± standard deviation (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 for the treatment groups compared to the control. #p < 0.05 and ##p < 0.01 for the BP exposure groups compared to the VP exposure groups at the same concentration. †p < 0.05 and ††p < 0.01 for comparison among the different concentrations in the same phosphorene nanomaterial exposure.

Collectively, exposure to environmentally relevant concentrations of BP and VP elicited oxidative stress responses in the algal cells. Li et al.²⁰ also reported that oxidative stress was the underlying cause of toxicity of BP to algae at higher doses (5 and 10 mg L⁻¹). Specifically, the phosphorus nanomaterials stimulated the production of intracellular ROS, primarily within the mitochondria. In response to the stress imposed by the phosphorus nanomaterials, the algal cells activated their antioxidant defense mechanisms and scavenged the ROS by modulating SOD activity, thereby protecting the cells from oxidative damage. As previously mentioned, exposure to BP did not induce growth-inhibitory toxicity towards algae, whereas the VP did induce toxicity (Fig. 2B). Therefore, although the BP and VP did not cause significant oxidative damage to algal cells in this study, they could exert toxic effects through other mechanisms, such as physical damage to the cell membrane, with the VP demonstrating more severe cytotoxicity. ROS-dependent oxidative stress and physical damage are proposed as the primary antibacterial mechanisms underlying the activity of VP and its exfoliated derivative.²² Zhang et al.³³ also concluded that, in addition to BP-induced ROS generation, other mechanisms may be involved, specifically physical damage to the cell membrane, which accounts for the cytotoxicity of BP. The sharp morphological characteristics of BP and VP nanosheets, as illustrated in Fig. 1A and B, resemble those of other 2D nanosheets and can easily penetrate the cellular membrane.³⁴ This penetration might result in internal cellular damage and disrupt interactions with the cytoskeletal framework. This possibility is supported by the results of the assessment of the interactions between the phosphorus nanomaterials and algal cells (Fig. 3G). Physicochemical characterization revealed that VP exhibited less aggregation and higher suspension stability than BP. This significantly enhanced its interaction with the cells and subsequent cellular uptake, ultimately leading to the observed increase of toxic effects. In addition, BP is unstable and could degrade to dissolved phosphorus acid which may be partially responsible to induce toxicity.¹² In the present study, the XPS analysis indicated that the solid VP might mainly contribute to the observed toxicity compared to the solid BP. This is also consistent with the previous conclusion that VP predominantly exhibited stronger toxic effects on T. obliquus than BP through a physical damage mechanism.

Effects of BP and VP on algal gene transcription

As far as we know, this is the first study to explore the impacts of BP and VP on the functional gene expression of algae at the RNA level. The gene expression distributions for the control group and the tested groups are illustrated in Fig. S3. The Venn diagram in Fig. 5A visually demonstrates that the number of genes shared among the BP, VP, and control groups was 13 916, significantly surpassing the number of unique genes in each group. Notably, the VP-treated group contained a greater number of unique genes (2326) compared to the control group (1152). In contrast, the BP-treated group had fewer unique genes (744) than the control group. The heatmap analysis of DEGs revealed significant differences between the algae exposed to VP and those exposed to BP (Fig. 5B). Compared to the control group, the number of both upregulated and downregulated DEGs in the algae exposed to VP was higher than in those exposed to BP (Fig. 5C and D). Specifically, there were 29 times more DEGs in the algae exposed to VP compared to those exposed to BP.

Fig. 5 Transcriptome analysis of T. obliquus exposed to the suspensions of BP and VP. (A): Venn diagrams showing overlap of gene expression between treatment and control groups. (B): Hierarchical clustering heatmap of DEGs. (C) and (D): Volcanic maps of DEGs for BP vs. control and VP vs. control respectively. Two vertical dotted lines in the volcanic maps distinguish DEGs that differed more than twofold, whereas DEGs above the horizontal dotted line had a P-value <0.05.

As illustrated in Fig. 6A, the DEGs in the algal cells exposed to BP primarily exhibited a ‘molecular function’ associated with ‘structural molecule activity’. Analysis of the ‘cellular component’ revealed that these DEGs were predominantly localized in the ‘ribosome’. Furthermore, these DEGs were primarily enriched in the ‘biological process’ of ‘ribosome biogenesis’. Similarly, as depicted in Fig. 6B, the DEGs in the algal cells exposed to VP also demonstrated a ‘molecular function’ related to ‘structural molecule activity’. The ‘cellular component’ analysis indicated that these DEGs were mainly localized in the ‘ribosome’. Additionally, these DEGs were enriched in the ‘biological processes’ of ‘ribosome biogenesis’ and ‘cell wall organization or biogenesis’. Fig. 6C and D present the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for both BP and VP, with a focus on the ribosome. This indicates that a group of DEGs primarily related to the ribosome, which served as the primary site of translation. While ribosomal proteins are primarily involved in the translation of mRNA, some ribosomal proteins may also participate in other cellular processes or respond to environmental stressors (e.g., pollutant-induced oxidative stress).³⁵ Furthermore, the involvement of ribosome-related DEGs might suggest several important physiological implications. For instance, when algal cells were exposed to a higher concentration of phosphorene nanomaterials, a decrease in the expression of ribosome biogenesis-related gene would be accompanied by a significant reduction in cell growth and photosynthesis, as indicated in Fig. 2.

Fig. 6 Transcriptome analysis of T. obliquus exposed to the suspensions of BP and VP. Statistical classification of GO annotation (A and B) and KEGG pathway enrichment analysis (C and D) of DEGs after exposure of T. obliquus (96 h) to BP and VP.

Changes in gene expression related to photosynthesis, ROS production, SOD activity, and mitochondrial membrane dynamics were analyzed in algae exposed to both BP and VP. As illustrated in Fig. S4A, exposure to VP resulted in significant alterations in a greater number of genes associated with photosynthesis compared to BP. Notably, the number of upregulated and downregulated DEGs induced by VP exposure was comparable. Fig. S4B shows that VP exposure caused significant changes in all genes related to ROS production, including two downregulated DEGs and four upregulated DEGs. Similarly, VP exposure induced significant changes in all genes associated with SOD activity, comprising two downregulated DEGs and one upregulated DEG (Fig. S4C). Furthermore, VP exposure led to significant changes in all genes related to the mitochondrial membrane, with all these DEGs exhibiting upregulation (Fig. S4D). Collectively, VP induced more marked alterations in functional DEG expression compared to BP. The differential effects of BP and VP exposure on functional DEG expression patterns were consistent with their distinct impacts on cellular biomarkers (i.e., photosynthetic pigments, ROS, SOD, and MMP). Therefore, there is reasonable evidence to suggest that these DEGs could participate in intercellular and intracellular signaling pathways, thereby influencing cellular physiological activities.

In addition, we also evaluated the correlation between the data obtained from the RNA-Seq and qRT-PCR for ten DEGs, as detailed in Fig. S5. We calculated a Pearson correlation coefficient of 0.918 between the results of RNA-Seq and qRT-PCR. Therefore, it is reasonable to conclude that the findings drawn from the transcriptome analysis were both reliable and accurate.

Effects of BP and VP on metabolism in algae

As shown in Fig. 7A, T. obliquus exposed to the BP exhibited a total of 264 differentially expressed metabolites (DEMs) compared to the control group, with 168 upregulated DEMs and 96 downregulated DEMs. Furthermore, the types of the TOP 20 upregulated and downregulated DEMs in the algae exposed to the BP are illustrated in Fig. 7B. As shown in Fig. 7C, T. obliquus exposed to the same concentration of VP exhibited a total of 556 DEMs compared to the control group, with 375 upregulated DEMs and 181 downregulated DEMs. Additionally, the types of the TOP 20 upregulated and downregulated DEMs in the algae exposed to the VP are presented in Fig. 7D.

Fig. 7 Metabonomics analysis of T. obliquus exposed to the suspensions of BP and VP. Volcanic map of DEMs for BP vs. control (A) and VP vs. control (C), in which two vertical dotted lines in the figure distinguish DEMs that differed more than twofold, whereas DEMs above the horizontal dotted line had a P-value <0.05. Names of the TOP 20 DEMs with up- or down-regulation for BP vs. control (B) and VP vs. control (D). Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001.

The KEGG pathway enrichment analysis of DEMs for the BP exposure (Fig. 8A) revealed that these pathways were mainly classified into ‘metabolism’, ‘genetic information processing’, and ‘environmental information processing’. Among the metabolism pathways, based on the proportion of DEMs enriched in each pathway to the total DEMs, the highest proportion is attributed to ‘global and overview maps’. Fig. 8B shows that the DEMs for the BP exposure were significantly enriched in three KEGG pathways: ‘cyanoamino acid metabolism’, ‘pyruvate metabolism’, ‘phenylalanine metabolism’, and ‘phenylpropanoid biosynthesis’.

Fig. 8 Metabonomics analysis of T. obliquus exposed to the suspensions of BP and VP. Classification of KEGG pathway of DEMs for BP vs. control (A) and VP vs. control (C). KEGG pathway enrichment analysis of TOP 20 DEMs for BP vs. control (B) and VP vs. control (D).

Similar to the BP, the KEGG pathway enrichment analysis of DEMs for the VP exposure also showed three mainly classified pathways (Fig. 8C), i.e., ‘metabolism’, ‘genetic information processing’, and ‘environmental information processing’. Similar to the BP, the ‘global and overview maps’ category also accounted for the highest proportion of DEMs in the case of VP exposure. However, unlike the BP exposure, an additional pathway within the genetic information processing category, namely ‘folding, sorting, and degradation’ was significantly enriched due to the VP exposure. Fig. 8D shows that the DEMs were significantly enriched in three KEGG pathways: ‘aminoacyl-tRNA biosynthesis’, ‘phenylalanine metabolism’, and ‘cyanoamino acid metabolism’.

Through comparative analysis, it became evident that exposure to both BP and VP significantly enriched the KEGG pathways of ‘phenylalanine metabolism’ and ‘cyanoamino acid metabolism’. Notably, ‘phenylalanine metabolism’ is a crucial source of phenolic antioxidant compounds. Meanwhile, ‘cyanoamino acid metabolism’ encompassed a series of essential biochemical processes that help maintain the water and electrolyte balance, regulate cellular metabolism, and generate specific bioactive substances. The KEGG pathways that are uniquely and significantly enriched under BP exposure include ‘pyruvate metabolism’ and ‘phenylpropanoid biosynthesis’. Pyruvate metabolism plays a pivotal role in maintaining cellular homeostasis and facilitating energy production.³⁶ In contrast, phenylpropanoids are essential for various physiological processes, enhancing plant tolerance and adaptability in stressful conditions.³⁷ In contrast to BP, the unique and significantly enriched pathway observed under VP exposure was ‘aminoacyl-tRNA biosynthesis’. Aminoacyl-tRNA molecules are essential within cells, playing a crucial role in protein synthesis by ensuring the accurate incorporation of amino acids into the growing polypeptide chain.³⁸

These results highlight that BP and VP exposures triggered distinct biological responses. While BP predominantly upregulates energy metabolism (specifically pyruvate metabolism) and phenylpropanoid-based defense systems, VP prioritized translational accuracy through aminoacyl-tRNA biosynthesis. Shared pathways, such as phenylalanine and cyanoamino acid metabolism, further indicated a coordinated strategy to combat oxidative stress and maintain metabolic equilibrium. Collectively, these adaptations suggested that BP and VP enhanced cellular robustness through complementary metabolic and biosynthetic reprogramming.

Integrated analysis of transcriptome and metabolome

Regarding the BP exposure, the absolute values of the correlation coefficients between the TOP 5 DEMs and the TOP 10 DEGs ranged from 0.700 to 0.990, as illustrated in Fig. 9A. It is estimated that 76% of these combinations exhibited correlation coefficients exceeding 0.80. Furthermore, a significant proportion, specifically 68%, demonstrated statistically significant correlations (p < 0.05), as shown in Fig. 9A. The KEGG pathways jointly enriched in the metabolomics and transcriptomics for the BP exposure, are depicted in Fig. 9B. ‘Pyruvate metabolism’ stood out as the most significantly enriched pathway, involving three DEMs namely one downregulated ‘phosphoenolpyruvic acid’ and two upregulated ‘acetyl-CoA’ and ‘succinic acid’, alongside one downregulated DEG (Table S3). ‘Phosphoenolpyruvic acid’ is a high energy phosphate compound involved in the glycolysis pathway.³⁹ Additionally, ‘phosphoenolpyruvic acid’ is situated at one end of the chloroplast pyruvate hub,⁴⁰ serving as a precursor of pyruvate or for the shiki-mate pathway.⁴¹ Furthermore, ‘phosphoenolpyruvic acid’ plays an important role in the photosynthetic reaction process in plants.⁴¹ The downregulation of ‘phosphoenolpyruvic acid’, might inhibit its associated metabolic pathways. Consequently, ‘phosphoenolpyruvic acid’ emerges as a key metabolite responsible for the metabolic toxicity effects observed in the algal cells induced by the BP exposure. Moreover, the molecular functions associated with the correspondingly downregulated DEGs encompassed ferric iron binding, phosphoenolpyruvate carboxykinase (ATP) activity, and ATP binding. The biological processes related to the DEGs encompassed the tricarboxylic acid cycle, gluconeogenesis, carbon utilization, as well as iron–sulfur cluster assembly.

Fig. 9 Interaction network of TOP 10 DEGs and TOP 5 DEMs for BP vs. control (A) and VP vs. control (C). Commonly enriched KEGG pathways in metabolomics and genomics for BP vs. control (B) and VP vs. control (D).

For the VP exposure, the correlation coefficients between the TOP 5 DEMs and the TOP 10 DEGs exhibited absolute values ranging from 0.958 to 0.998, as illustrated in Fig. 9C. Impressively, all combinations (100%) showed significant correlations (p < 0.05). The KEGG pathways jointly enriched by the metabolomics and transcriptomics for the VP exposure are presented in Fig. 9D. The most significantly enriched pathway was identified as ‘aminoacyl-tRNA biosynthesis’. Within the ‘aminoacyl-tRNA biosynthesis’ pathway, one downregulated DEM (10-formyl-THF) and nine upregulated DEMs, namely L-(−)-methionine, L-tryptophan, L-lysine, L-arginine, threonine, L-phenylalanine, L-tyrosine, L-glutamic acid, and L-aspartic acid, are involved (Table S4). 10-Formyl-THF plays a crucial role in de novo purine biosynthesis,⁴² which is a pathway for synthesizing purine nucleotides that are essential for numerous biological processes, particularly nucleic acid replication.⁴³ Consequently, ‘10-formyl-THF’ had emerged as a pivotal metabolite that was responsible for the metabolic toxicity effects observed in algal cells following exposure to VP. Moreover, the ‘aminoacyl-tRNA biosynthesis’ pathway included 3 downregulated DEGs and 15 upregulated DEGs (Table S4). Among the downregulated DEGs, the most significantly downregulated gene was associated with a range of molecular functions, including sarcosine oxidase activity, aminoacyl-tRNA ligase activity, ATP binding, transmembrane transporter activity, lysine-tRNA ligase activity, nucleotide binding, and cysteine-tRNA ligase activity. These functions predominantly occurred at the membrane or cytoplasm loci. Furthermore, this DEG was involved in various biological processes, such as the cysteine metabolic process, lysyl-tRNA aminoacylation, tetrahydrofolate metabolic process, tRNA aminoacylation for protein translation, lysine biosynthetic process, cysteinyl-tRNA aminoacylation, glycine metabolic process, threonine metabolic process, L-serine metabolic process, additional tRNA aminoacylation processes, and transmembrane transport. Overall, the effect mechanisms of BP and VP on T. obliquus at a transcriptomic and metabolomic level were found to be distinctly different.

Summary and implications

As the application prospects of phosphorene nanomaterials such as BP and VP nanosheets continue to expand, it is essential to possess knowledge regarding their potential environmental impacts. Our study findings highlight significant disparities in the toxicological effects of BP and VP nanosheets at environmentally relevant concentrations towards the freshwater microalgae T. obliquus, spanning from molecular and cellular levels up to the population level. VP exhibited a relatively high dispersion stability and low degradation and induced stronger growth-inhibitory toxicity towards the algae compared to BP. This difference in the population toxicity might be attributed to the relatively severe physical damage inflicted by VP on the algal cell membranes. Furthermore, both BP and VP induced cellular oxidative stress responses, yet neither caused oxidative damage to the cells. Multi-omics technologies have elucidated key pathways, critical metabolites, and driving genes associated with the molecular-level differences in the toxicological effects of BP and VP. These pathways could enhance our understanding of the transformation and excretion processes of various phosphorene nanomaterials within organisms. Additionally, specific metabolites such as phosphoenolpyruvic acid and 10-formyl-THF and their associated genes would serve as biomarkers for monitoring potential environmental contamination by diverse phosphorene nanomaterials, thereby providing robust support for environmental monitoring and early warning systems related to these materials. In summary, this contribution represents a solid step forward in the comprehensive assessment of the environmental risks posed by phosphorene nanomaterials.

Author contributions

Haoxiang Zhang: investigation, data curation, formal analysis, visualization, writing-original draft. Fan Zhang: conceptualization, formal analysis, visualization, writing – review & editing, funding acquisition, project administration. Zhuang Wang: conceptualization, resources, investigation, methodology, data curation, formal analysis, visualization, writing – review & editing, supervision, funding acquisition, project administration. Willie J. G. M. Peijnenburg: conceptualization, formal analysis, writing – review & editing.

Conflicts of interest

There are no conflicts to declare.

Data availability

Supplementary information: additional supporting information, including supplementary tables and figures, is included as part of the supplementary information (SI). See DOI: https://doi.org/10.1039/D5EN00302D.

The data supporting this article have been included in the main text as well as in the SI.

Acknowledgements

The research described in this work was supported by the National Natural Science Foundation of China (grant number 31971522) and by the Scientific Research Foundation for Young Hundred-Talents Program of Yangzhou University (grant number 137013547). We also thank the reviewers for their valuable comments on the manuscript.

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