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

## TL;DR

This study examines how imidacloprid and microplastics together affect tadpoles, showing growth inhibition and microbiome disruption.

## Contribution

A new multi-tiered framework is proposed to assess combined pollutant effects on amphibians.

## Key findings

- Combined exposure to imidacloprid and microplastics inhibited tadpole growth and caused oxidative stress.
- Exposure increased nuclear anomalies and disrupted gut/skin microbiota composition.
- A multi-tiered assessment framework was developed to evaluate compound pollutant toxicity.

## Abstract

Q: How did IMI + MPs affect the tadpole LC50/TK parameters compared to their impact on growth? A: IMI + MPs negligibly affected the tadpole LC50/TK parameters compared to growth.

Q: What was the difference in nuclear anomaly rates between tadpoles exposed to IMI alone and those exposed to IMI + MPs? A: IMI + MPs increased the nuclear anomaly rates in tadpoles compared to IMI.

Q: How did the combination of IMI+ MPs influence the gut and skin microbiota in tadpoles? A: IMI + MPs induced a gut/skin microbiota diversity imbalance in tadpoles.

Q: What was developed to assess the integrated effects of pollutants like IMI + MPs? A: A framework for a new multifaceted response evaluation was established to assess integrated effects.

This study investigated the individual and combined effects of imidacloprid and polyethylene microplastics on Hoplobatrachus rugulosus tadpoles, focusing on acute toxicity, growth, oxidative stress, and gut/skin microbiota. The results showed that while microplastics did not significantly alter the acute toxicity of imidacloprid, combined exposure markedly inhibited tadpole growth and induced oxidative stress and genotoxic damage. Furthermore, significant shifts in microbial community composition were observed, particularly in the gut and skin, with imbalances in dominant bacterial taxa. This study proposes a multi-tiered assessment framework to evaluate the combined toxicity of pollutants, providing new insights into their ecological risks. The findings highlight the need for the prudent use of pesticides and agricultural plastics to safeguard ecosystem health.

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.