Exposure to Rice Straw Ash Alters Survival, Development and Microbial Diversity in Amphibian Tadpoles

Abstract

Amphibians are increasingly threatened by human activities, with rice straw burning emerging as a significant yet underexplored hazard. This practice may release harmful polycyclic aromatic hydrocarbons (PAHs), disrupt ecosystems, and affect amphibians. However, the impact on tadpole microbiota and development remains unclear. This study used scanning electron microscopy (SEM) and chemical analysis to characterize straw ash toxicity, assessed rice straw aqueous extracts of ash (AEA; 0, 0.75, 1.5, 3, and 6 g L^-1) on Rana dybowskii tadpoles survival, growth, and development, and analyzed skin and gut microbiota via Illumina sequencing. Within the AEA, 10 varieties of PAHs exhibited higher quantities, including acenaphthylene, acenaphthene, and anthracene. SEM revealed irregular, porous, layered ash particles. Higher AEA concentrations reduced survival, delayed development, and affected body mass. The alpha diversity of both skin and gut microbiota significantly varied among groups. Beta diversity analyses indicated substantial shifts in microbial community structure with increased AEA concentrations. Linear discriminant analysis (LEfSe) identified microbial taxa enrichment and shifts, including the increase of potentially pathogenic genera such as Citrobacter and Yersinia in high-concentration groups. BugBase analysis showed significant phenotypic changes in microbial communities. Our findings expose rice straw ash as a silent, global toxin that disrupts amphibian microbiota, growth, and survival-redefining routine straw burning as a planetary biodiversity hazard and urging immediate, sustainable reforms to protect wetland ecosystems.

Keywords: ecological conservation; ecosystem stability; microbial diversity; microbiota; straw burning.

FIGURE 1

Scanning electron microscopy (SEM) analysis of straw ash. SEM reveals the post‐combustion ash particle morphology and structure at varying magnifications. At a 500‐fold magnification, (A and B) illustrate the overall morphology and surface textures of two separate particle assemblies. At a 10,000‐fold magnification, (C and D) provide a comprehensive analysis of the microstructural complexities of a solitary particle aggregate.

FIGURE 2

Differences in body mass, survival, and development stage among five treatment groups. Significant differences in body mass (A), survival (B), and developmental stage (C) among the five groups (S0, S0_75, S1_5, S3, and S6). Panel C presents the developmental progress of R. dybowskii tadpoles exposed to rice straw ash aqueous extracts for 28 days, evaluated based on the Gosner staging system. The developmental stage of five groups is expressed as the percentage of development in G22‐23, while others belong to G24‐G25 (n = 35).

FIGURE 3

The alpha diversity of R. dybowskii tadpoles' skin and gut microbiota in five treatment groups. A comparative analysis of skin (A) and gut (B) microbiota alpha diversity in tadpoles exposed to five treatment concentrations (SC0, SG0, SG3: N = 9, SC0_75, SC1_5, SC3, SC6, SG0_75, SG1_5, SG6: N = 8) straw ash conditions. Analyses utilized ACE, Chao, Shannon, and Sobs indices. Significance levels are indicated: *, **, and *** for p values falling in 0.01 < p < 0.05, 0.001 < p < 0.01, and p < 0.001, respectively.

FIGURE 4

Assessing aqueous extracts of rice straw ash impact on beta diversity of tadpole microbiota with non‐metric multidimensional scaling (NMDS). To analyse the impact of aqueous extracts of rice straw ash at different concentrations on community structure and dispersion, data at the OTU level were utilized, employing the Bray–Curtis dissimilarity index (A and C) and weighing UniFrac distance (B and D). NMDS plots illustrate samples from the skin (A and B) and gut (C and D).

FIGURE 5

Characteristics of tadpole skin and gut microbiome composition under five treatment aqueous extracts of rice straw ash concentration levels. This study utilized bar charts to analyze microbiota at the phylum (A and C) and genus (B and D) levels in skin (A and C) and gut (B and D) microbiota among five concentrations (SC0, SG0, SG3: N = 9, SC0_75, SC1_5, SC3, SC6, SG0_75, SG1_5, SG6: N = 8). The charts only display phyla and genera with a relative abundance of over 1% in at least one sample.

FIGURE 6

Investigating variations in tadpole skin and gut microbiota under five treatments of aqueous extracts of rice straw ash exposure using linear discriminant analysis effect size (LEfSe). The cladogram delineates the phylogenetic distinctions among microbiota taxa, highlighting the divergences in skin (A) and gut (B) microbiota among five concentrations (SC0, SG0, and SG3: N = 9; SC0_75, SC1_5, SC3, SC6, SG0_75, SG1_5, and SG6: N = 8). Color differentiation post‐treatment grouped samples into distinct categories. The relative abundance of each taxon is indicated by the size of the circles within the cladogram. Circle size denotes group abundances, while a detailed multiclass analysis reveals discrepancies across at least one class, delineating the taxonomic hierarchy from domain to genus via concentric circles. Labels for phylum through genus and taxa with LDA scores exceeding 4 are illustrated. The color‐coded legend on the right indicates the taxonomic classification of each node, ranging from phylum (p) to genus (g), and corresponds to the colors represented in the cladogram.

FIGURE 7

Using BugBase examined aqueous extracts of rice straw ash impact on R. dybowskii skin and gut microbiomes at various concentrations. Assessing the impact of aqueous extracts of rice straw ash at five concentrations (SC0, SG0, and SG3: n = 9; SC0_75, SC1_5, SC3, SC6, SG0_75, SG1_5, and SG6: n = 8) on the phenotype of skin (A) and gut (B) microbiota in tadpoles, using the BugBase approach to examine and predict bacterial characteristics. Significance levels are indicated: *, **, and *** for p values falling in 0.01 < p < 0.05, 0.001 < p < 0.01, and p < 0.001, respectively.