Abstract
The concerns about the presence of microplastics (MPs) in marine ecosystems have widely increased in the past years. This is reflected in a growing number of studies addressing the effects of exposure to these materials in indigenous, farmed and even laboratory marine animals subjected to toxicity-oriented bioassays. There have been, however, many constraints in the detection of MPs in biological tissues, as routine histological techniques tend to degrade these materials, which are especially sensitive to organic solvents. This issue hinders the application of standard histopathological procedures based on convenient paraffin wax-embedding protocols, with consequences for biomonitoring and bioassay procedures. The method described here was developed and validated for the detection of polystyrene microplastics in biological tissue processed for paraffin-based histology. The strategy was developed and tested from whole-soft body sections of marine mussels that internalised the MPs following dedicated bioassays. The protocol is based on the replacement of xylenes with isopropanol for the purpose of intermediate infiltration and deparaffinization. Special modifications for staining, mounting and archiving are needed and are detailed as well. The protocol is shown to be a highly cost- and time-effective procedure compatible with formalin-based fixatives plus standard sectioning and staining, yielding complete preservation of MPs and optimal tissue conditioning. The method also produced excellent results with pre-stained MPs, with fluorochromes included, altogether providing excellent localisation of polystyrene MPs in paraffin-processed biological tissue.
Highlights
The ubiquity of plastic debris in marine ecosystems has been leading to a growing awareness towards these emerging potential pollutants in the past years, with emphasis on their potential internalisation by aquatic organisms
Particle fluorescence was not affected by this solvent, showing that the protocol was compatible with chromogens and fluorochromes embedded in plastic, which are handy modifications for localising MPs (Fig. 1d)
The proposed methodology is suitable and expeditious for studies involving the search for microplastics within biological tissues
Summary
The ubiquity of plastic debris in marine ecosystems has been leading to a growing awareness towards these emerging potential pollutants in the past years, with emphasis on their potential internalisation by aquatic organisms. Despite the wide variety of plastics being released into the marine environment, polystyrene is considered to be one of the most important polymers (Hidalgo-Ruz et al 2012; Moore 2008). Detecting microplastics (MPs) eventually ingested by organisms is a key component of both biomonitoring and testing procedures. Histopathology, in particular, is, as for other contaminants, a critically important tool that permits inferring the presence of MPs in tissues and organs and eventual adverse effects altogether. Standard histological processing has been shown to be incompatible with MPs, polystyrene and similar polymers in particular, as strong organic solvents tend to dissolve the materials or render them too brittle for proper sectioning. Previous experiments conducted by the authors of the present work showed that solvents such as xylenes or chloroform and even alcohols damage, deform or completely dissolve
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