# Standardized trimodal histopathological examination for microplastic detection and tissue-level assessment in green mussels (Perna viridis) cultivated near an industrial estate in Rayong, Thailand

## TL;DR

This study develops a new method to detect microplastics in green mussels using three microscopy techniques and finds that digestive tracts are most affected.

## Contribution

A standardized trimodal histopathological method for microplastic detection in green mussels is developed and validated.

## Key findings

- Digestive tracts showed the highest microplastic accumulation (64.1%) in green mussels.
- Most microplastics were irregular fragments (91.67%) sized 10–100 μm.
- Only 11.1% of microplastic-positive tissues showed mild to moderate histopathological lesions.

## Abstract

Microplastics (MPs) are persistent pollutants that build up in filter-feeding marine animals. Green mussels (Perna viridis), commonly eaten in Thailand, are effective bioindicators of water pollution. However, there are still limited standardized histopathological methods for detecting MPs and assessing lesions. This study aimed to develop a three-part histopathological exam using light microscopy, polarized light microscopy, and Nile Red (NR) fluorescence, and to analyze MP distribution and linked tissue effects in mussels collected from industrial shoreline areas in Rayong Province.

Sixty-one mussels were collected from three coastal sites near an industrial estate. Target tissues were processed using a novel isopropanol-based, xylene-free protocol to prevent MP loss. Serial sections were stained with hematoxylin–eosin, evaluated under polarized light, and analyzed for NR fluorescence. Particle confirmation required co-localization across all three methods. Histopathological lesions were scored semi-quantitatively, and statistical associations were assessed using Fisher’s exact test, McNemar’s test, and generalized estimating equations.

MP prevalence was 59.0% using light and polarized microscopy, 44.3% with NR fluorescence, and 39.3% with trimodal confirmation. The digestive tract showed the highest MP accumulation (64.1%), followed by the gills (33.3%) and the digestive glands (15.4%). Most MPs were irregular fragments (91.67%), mainly 10–100 μm in size. Mussel size was not significantly linked to MP contamination (p = 0.224). Notably, 88.9% of MP-positive tissues showed no observable histopathological changes; only 11.1% had mild to moderate lesions, including epithelial cell damage and hemocyte infiltration in digestive tracts, gills, and digestive glands.

The standardized trimodal histopathological approach offers a reliable, fast, and xylene-free method for MP detection in P. viridis. Digestive tracts, gills, and digestive glands are the most informative tissues for biomonitoring. Although many tissues did not show obvious lesions, the presence of mild pathological changes highlights the potential for sublethal effects in chronically exposed populations. This method improves diagnostic accuracy by reducing false positives and provides a consistent framework for MP surveillance in industrial coastal zones.