Titanium carbide (Ti₃C₂T_x) nanomaterials were purchased from Xianfeng Nano Technology Co., Ltd., Nanjing, China. The size and morphology of Ti₃C₂T_x were characterized using a transmission electron microscope (JIM-2100, Japan). The charge and hydrated size of Ti₃C₂T_x were determined using a Bru -Kehaven high-sensitivity Zeta potential and size analysis instruments (NanoBrook 90plus PALS, United States).

Daphnia magna was procured from the Guangdong Provincial Laboratory Animal Inspection Institute. Daphnia were continuously cultured for three generations in beakers containing tap water aerated for over 3 days within an artificial climate chamber (Shanghai Yiheng Scientific Instrument Co., Ltd.). Cultivation conditions were set as follows: light–dark cycle 16 h:8 h, temperature maintained at 21 ± 1 °C. Daphnia were fed twice daily with Chlorella vulgaris, at a concentration of 1 × 10⁵ cells/mL. These cultivation conditions comply with the protocol provided by the International Organization for Standardization (ISO 6341: 2012).

This experiment established three Ti₃C₂T_x treatment groups: 0 mg/L (control group), 0.01 mg/L, and 1 mg/L. These concentrations were selected based on environmentally relevant titanium concentrations (Kaegi et al., 2008). To investigate the growth and development of D. magna, 1-day-old neonates (≤24 h) were selected because they are in the early developmental stage with high sensitivity to environmental pollutants, and their growth and development processes are relatively homogeneous, which can reduce the experimental variation caused by individual differences. 1-day-old neonates were exposed to three Ti₃C₂T_x treatment groups, with 5 individuals per 50 mL of exposure solution. The exposure period lasted for 7 days, during which Chlorella was fed daily at a concentration of 1 × 10⁵ cells/mL, and the exposure solution was completely renewed every 2 days. After the exposure period, D. magna was subjected to growth and development phenotypic analysis. Meanwhile, Daphnia samples were collected and stored in a − 80 °C ultra-low temperature refrigerator for subsequent analysis of the expression of growth and development-related genes. In addition, to obtain sufficient samples of the intestinal microbiota of D. magna, 14-day-old individuals with a stable gut microbiome were exposed following the same procedure described above. To avoid the interference of secondary changes in the gut microbiome caused by long-term growth inhibition, the daphnids were dissected under a stereomicroscope in a sterile environment after 1 day of exposure, and the intestines from every 20 individuals were pooled as one sample for intestinal microbiota analysis.

The accumulation of Ti₃C₂T_x within D. magna was observed and analyzed following the methods described in previous research (Xiang et al., 2024). Ti₃C₂T_x, being a black substance, is readily observable and detectable within the transparent bodies of D. magna. Consequently, this study employed a standard optical microscope to observe Ti₃C₂T_x within D. magna. Briefly, Daphnia exposed for 7 days were absorbed onto concave glass slides and covered with coverslips. Samples were then placed under an Olympus optical microscope (Model BX53F2C) to observe Ti₃C₂T_x accumulation within the organisms. Distribution within the intestinal tract was documented using a CCD-D23 camera.

The growth and developmental phenotypes of D. magna were analyzed with reference to the methods reported in previous study (Qi et al., 2022). The primary biological indicators for growth and development in this study comprised moulting frequency, body length, body width, and absolute growth rate. Briefly, to determine the moulting frequency, Daphnia were observed and counted under a microscope every 24 h during the exposure period to record the number of molts. Following the conclusion of the 7-day exposure period, Daphnia were examined under an Olympus optical microscope (Model BX53F2C), with changes in body length and width documented and analyzed via CCD-D23 imaging. Concurrently, the absolute growth rate of Daphnia was analyzed based on body length data.

The expression of growth and development-related genes in D. magna was analyzed with reference to the method reported in previous study (Chen et al., 2021). To evaluate growth and development progression, mRNA expression was measured for cyp314, cyp18a1, ecra, ecrb, usp., hr3, ftz-f1, and cpa1 genes, which are critical regulators of D. magna growth and development. Briefly, total RNA was extracted from D. magna using the Trizol reagent kit according to the manufacturer’s protocol. Subsequently, the concentration and integrity of total RNA in each sample were analyzed using a nucleic acid and protein analyzer (NanoDrop2000) and agarose gel electrophoresis, respectively (Supplementary Figure S1; Supplementary Table S1). Finally, RNA was reverse transcribed into cDNA for quantitative real-time PCR detection. Primers for amplifying and detecting relevant genes in D. magna used in this study are provided in the Supplementary Table S2.

Real-time quantitative PCR (RT-qPCR) was analyzed using an ABI 7300 real-time quantitative PCR instrument (Applied Biosystems, United States). Briefly, First, the Daphnia cDNA obtained via reverse transcription was combined with primers upstream and downstream of the target gene and internal control gene. Subsequently, a 10 μL PCR reaction system was prepared using 2 × ChamQ SYBR Color qPCR Master Mix and sterile water following the SYBR Green I dye method. Reaction conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles comprising 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 40 s. A melting curve analysis was subsequently performed to confirm reaction specificity. Finally, the β-actin gene from D. magna served as the reference internal control gene, and the relative mRNA expression levels of the target genes were calculated using the 2^-ΔΔCt method.

The analysis of the intestinal microbiota in D. magna was performed via high-throughput sequencing with reference to the method reported in previous study (Li et al., 2022). Briefly, DNA was first extracted from Daphnia intestinal samples using a DNA extraction kit (Omega Bio-tek, Norcross, GA, United States). Sample DNA integrity and concentration were assessed via 1% agarose gel electrophoresis and NanoDrop2000 (Thermo Scientific, United States), respectively (Supplementary Figure S1; Supplementary Table S3). Secondly, using the extracted DNA as template, PCR amplification of the 16S rRNA gene was performed with primers 341F (5’-CCTACGGGNGGCWGCAG-3′) and 785R (5’-GACTACHVGGGTATCTAATCC-3′). Subsequently, PCR products were recovered and purified via 2% agarose gel electrophoresis. Finally, recovered products were quantified using the Qubit 4.0 system (Thermo Fisher Scientific, United States) and sequenced on Shanghai Meiji Biotechnology Co., Ltd.’s Illumina PE300/PE250 platform.

Following sequencing completion, Operational Taxonomic Unit (OTU) clustering analysis was performed on quality-controlled, assembled sequences using 97% similarity. Concurrently, microbial community diversity indices were analyzed based on OTU data. Subsequently, OTU taxonomic annotation was performed using the Silva 16S rRNA gene database (v138) at a 70% confidence threshold, with community composition at various taxonomic levels quantified for each sample. Finally, functional prediction of microbial communities was analyzed using Tax4Fun. All microbial data analyses in this study were conducted on the Shanghai Meiji Bio Cloud Platform.¹

The data from this study underwent normality testing via the Kolmogorov–Smirnov method. To distinguish significant differences between the control and treatment groups, a one-way analysis of variance (ANOVA) was performed using SPSS 26.0 software, followed by post-hoc multiple comparisons (Duncan’s test). A p-value < 0.05 indicates statistically significant differences between the control and treatment groups.

Electron microscopy characterization revealed (Supplementary Figure S3) that the Ti₃C₂T_x exhibited the following typical features: the Ti₃C₂T_x material displayed distinct irregular flakes with a diameter of approximately 100 nm, showing no significant large-area agglomeration (Supplementary Figure S3A). The surface charge of Ti₃C₂T_x was negatively charged at approximately −18 mV, with a hydrated sheet diameter of approximately 1.8 μm. Furthermore, optical microscopy revealed (Supplementary Figure S3) that exposure to Ti₃C₂T_x can lead to their accumulation in Daphnia. No Ti₃C₂T_x was observed within the bodies of D. magna in the control group (Supplementary Figure S3B). However, Ti₃C₂T_x accumulation was observed in D. magna following exposure to both 0.01 and 1 mg/L concentrations, with the primary distribution occurring within intestinal tissues (Supplementary Figures S3C,D).

Exposure to Ti₃C₂T_x can induce abnormal growth phenotypes in D. magna (Figure 1). Compared with the control group, exposure to 0.01 and 1 mg/L Ti₃C₂T_x significantly reduced the moulting frequency of D. magna. During the 7-day exposure period, the average moulting frequency per individual in the control group was 5.15 ± 0.19, while that in the 1 mg/L Ti₃C₂T_x group was 4.7 ± 0.26 (Figure 1A). Compared with the control group, exposure to both 0.01 and 1 mg/L Ti₃C₂T_x induced a significant reduction in the body length and width of D. magna. After 7 days of exposure, the average body length and width per individual in the control group were 2.37 ± 0.03 mm and 1.68 ± 0.04 mm, respectively, while those in the 1 mg/L Ti₃C₂T_x group were 2.09 ± 0.05 mm and 1.44 ± 0.05 mm (Figures 1B,C). Compared with the control group, exposure to 0.01 and 1 mg/L Ti₃C₂T_x both significantly reduced the absolute growth rate of D. magna. After 7 days of exposure, the average absolute growth rate in the control group was 0.21 ± 0.004 mm/d, while that in the 1 mg/L Ti₃C₂T_x group was 0.17 ± 0.007 mm/d (Figure 1D).

Exposure to Ti₃C₂T_x induced differential expression of genes related to growth and development in D. magna (Figure 2). Compared with the control group, exposure to 0.01 mg/L Ti₃C₂T_x significantly increased the expression of the growth and development genes cyp314 and ftz-f1 in D. magna, while significantly decreasing the expression of the genes ecra and hr3 (Figures 2A,G,C,F). Notably, exposure to 1 mg/L Ti₃C₂T_x induced a significant reduction in the expression of the D. magna developmental genes cyp18a1, ecra, usp., hr3, and cpa1, while having no apparent effect on the expression of the genes ecrb and ftz-f1 (Figures 2B–H). Exposure to both 0.01 and 1 mg/L Ti₃C₂T_x induced a significant reduction in expression of the D. magna developmental genes ecra and hr3.

Exposure to high concentrations of Ti₃C₂T_x showed a trend toward increased diversity and richness of the intestinal microbial community in D. magna (Figure 3). Compared to the control group, exposure to 0.01 mg/L Ti₃C₂T_x had no significant effect on Ace and Chao indices, whereas 1 mg/L exposure showed a trend toward increased Ace and Chao indices (Figures 3A,B). Similarly, exposure to 0.01 mg/L Ti₃C₂T_x showed no significant effect on the Shannon index, whereas 1 mg/L Ti₃C₂T_x exposure exhibited an increasing trend in the Shannon index (Figures 3C,D). Notably, exposure to 0.01 mg/L Ti₃C₂T_x resulted in a slight decrease in the Simpson index, whereas exposure to 1 mg/L Ti₃C₂T_x caused a significant reduction in the Simpson index.

Exposure to Ti₃C₂T_x induced significant alterations in the phylum-level microbial communities of the intestinal tract in D. magna (Figure 4). Principal coordinate analysis (PCoA) results revealed (Figure 4A) that the first principal component accounted for 46.1% of variance, while the second component contributed 33.43%, indicating strong discriminative power between treatment groups. The Venn diagram results (Figure 4B) revealed 745, 960, and 1,656 OTUs in the control, 0.01 mg/L, and 1 mg/L Ti₃C₂T_x groups, respectively. Among these OTUs, 365 were shared among the different treatment groups (control group, 0.01 mg/L, and 1 mg/L), accounting for 16.37% of the total OTUs. In addition, the number of unique OTUs in the 0.01 mg/L Ti₃C₂T_x group was 337 (15.11%), while that in the 1 mg/L group was 991 (44.44%). Results at the microbial phylum level (Figure 4C) showed that in the control group, the most abundant phylum in the intestinal microbiota was Proteobacteria (71.94%), followed by Bacteroidota (18.35%), Firmicutes (6.53%), and Actinobacteriota (1.72%). In the 0.01 mg/L Ti₃C₂T_x group, Proteobacteria was still the dominant phylum (64.19%), followed by Bacteroidota (21.20%), Firmicutes (11.52%), and Actinobacteriota (1.31%). For the 1 mg/L Ti₃C₂T_x group, the phylum-level abundance ranking was Proteobacteria (56.71%), Bacteroidota (23.87%), Firmicutes (11.64%), and Actinobacteriota (3.90%).

Exposure to Ti₃C₂T_x induced significant changes in the intestinal microbiota community of D. magna at the genus level (Supplementary Figure S4). Compared with the control group, 0.01 mg/L Ti₃C₂T_x decreased the abundances of Pseudomonas, Aeromonas, and Rhodobacteraceae in the intestines of D. magna, while increasing the abundances of Pedobacter and Bacillus. In contrast, 1.00 mg/L Ti₃C₂T_x reduced the abundances of Blastomonas, Pseudomonas, Aeromonas, and Chitinophagales, and elevated the abundances of Rhodobacteraceae, Bacillus, Acinetobacter, and Phascolarctobacterium in the Daphnids’ intestines (Supplementary Figure S4).

Predictions of gut microbial functions indicated that Ti₃C₂T_x exposure induced significant differences in the abundance of gut microbial functional groups associated with the growth and development of D. magna (Figure 5). Among the functional categories directly related to the growth and development of D. magna, exposure to 0.01 and 1 mg/L Ti₃C₂T_x both decreased the abundances of amino acid transport and metabolism, energy production and conversion, and posttranslational modification, protein turnover, chaperones. Exposure to 0.01 mg/L Ti₃C₂T_x significantly reduced the abundances of inorganic ion transport and metabolism, replication, recombination and repair, cell membrane biogenesis, carbohydrate transport and metabolism, and nucleotide transport and metabolism (Figure 5A). For the functional categories indirectly associated with D. magna growth and development, exposure to 0.01 and 1 mg/L Ti₃C₂T_x both decreased the abundance of the cell motility functional category and increased that of the cytoskeleton functional category (Figure 5B).