Bioaccumulation, Ecotoxicity, and Microbial Responses in Hoplobatrachus rugulosus Tadpoles Following Co-Exposure to Imidacloprid and Microplastics

Abstract

Agricultural organic pollutants have been identified as a key factor contributing to amphibian population decline, particularly during early developmental stages when tadpoles are frequently exposed to neonicotinoids (NEOs) and microplastics (MPs). In this study, Hoplobatrachus rugulosus tadpoles were exposed to imidacloprid (IMI: 0.045, 0.45, and 4.5 mg L^-1) and polyethylene-derived MPs (10 mg L^-1) from agricultural mulch films, both individually and in combination. We systematically evaluated acute toxicity, bioaccumulation, developmental and oxidative stress responses, and changes in the skin and gut microbiota. The results showed that the 96 h median lethal concentration (LC₅₀) of IMI was 44.8 mg L^-1 in the IMI-only group and was 40.5 mg L^-1 in the IMI + MPs group, indicating the negligible impact of MPs on acute toxicity. However, in the highest co-exposure group (IMI4.5 + MPs), tadpole body length and weight decreased by 14.7% and 22.6%, respectively, alongside marked changes in oxidative stress, whereby catalase (CAT) and superoxide dismutase (SOD) activities were suppressed, while malondialdehyde (MDA) levels increased by 35%, indicating elevated lipid peroxidation. Furthermore, the micronucleus frequency in erythrocytes was significantly elevated, suggesting genotoxic effects. Microbial community analysis revealed significant shifts in the relative abundance of gut and skin microbiota under IMI + MPs exposure, with a notable enrichment of Proteobacteria, Fusarium, Actinomycetota, and Bacteroidota, indicating the disruption of host-microbiome interactions. This study proposes a comprehensive multi-tiered assessment framework encompassing environmental exposure, bioaccumulation, toxicological endpoints, oxidative stress biomarkers, and microbiome shifts. Our findings provide new mechanistic insights and quantitative evidence on the compound threats posed by IMI and MPs to amphibians in aquatic environments.

Keywords: combined toxicity; imidacloprid; microbial diversity; microplastic; tadpole; toxicokinetics.

Figure 1

Morphological parameters or changes observed in H. rugulosus tadpoles exposed to IMI in the absence or presence of MPs after 28 days of exposure. Average micronucleus rate and average nuclear anomaly rate of IMI and MPs under single and combined exposure of Hoplobatrachus rugulosus tadpoles. (A) Body weight for IMI; (B) body length for IMI; (C) average micronucleus rate; (D) average nuclear anomaly rate. All weights are the fresh weight of tadpoles (p < 0.05, D and LSD test). Values represent the mean ± SD. Different lowercase letters above each bar denoted significant differences between treatments (p < 0.05; n = 6 animals/group).

Figure 2

The influence of IMI on the biochemical parameters in tadpoles. (A) AChE activity. (B) CAT activity. (C) SOD activity. (D) MDA content. All weights are the fresh tadpole weight (p < 0.05, D and LSD tests). Values represent the mean ± SD. Different lowercase letters above each bar denoted significant differences between different treatments (p < 0.05).

Figure 3

Results of integrated biomarker responses index (IBRv2) calculations based on growth/development, biochemical parameters, and erythrocyte nuclear abnormalities. Star plots for MPs, IMI0.045, IMI0.45, IMI4.5, IMI0.045 + MPs, IMI0.45 + MPs, and IMI4.5 + MPs on 7 (A), 14 (B), 21 (C), and 28 days (D).

Figure 4

Differences or similarities in the microbial community composition between different IMI treatment groups ((A1–C1) represents bacteria; (A2–C2) represents fungi) in the absence and presence of MPs after 28 days of exposure. (A) Hierarchical clustering of Bray–Curtis distance generated according to the operational taxonomic unit (OTU) of the tadpole microbiome. (B) Principal coordinate analysis (PCoA) indicating differences in the gut microbiome between CK and NEO or individual MP groups. (C) The Venn diagrams indicate the number of unique bacterial OTUs in the tadpole gut microbiota among individual and combined treatments. The blue font represents the gut, while the red font represents the epidermis.

Figure 5

Flow diagram of simplified multifaceted evaluation framework for the combined toxicity of an NEO and MPs to aquatic tadpoles. CAT: catalase; AChE: acetylcholinesterase; MDA: malondialdehyde; SOD: superoxide dismutase; k_u: uptake rate constant; k_e: elimination rate constant (K_e); BCF: bioconcentration factor. (A) Identification of target organic pollutants; (B) Identification of aquatic organisms; (C1) Bioaccumulation of organic pollutants in tadpoles; (C2) Physiology, biochemistry and gut microbial response to pesticides in tadpoles; (D1) Data access: uptake process, elimination process; (D2) Data access: Growth and development, biochemical indicators; (D3) Data access: Gut/epidermis and microorganisms; (E1) Data analysis: Toxicokinetics, toxicodynamics and residual time estimation; (E2) Data analysis: Histological observations, effect-dose analysis and response index; (E3) Data analysis: Microbial diversity index; (F1) Parameter characterization; (F2) (F3) Response sensitivity indicator screening; (G1) Explanation of the meaning of parameter changes; (G2) (G3) Complex toxicity interaction mode; (H) Analysis of impact mechanisms.