Improvements in histological technique for the ecotoxicological assessment using small biological samples

Abstract Histological techniques consist of traditional and very important tools for a better understanding of cellular morphology and physiology in ecotoxicological studies. Despite bioassays generally recommending the use of small test organisms, based on some advantages as their low maintenance costs and large replicates’ number, the histological processing of small organisms is not commonly used due to demanding laboratorial handling. The present study proposes an accessible protocol for a histological method in fish larvae with a maximum length of 8 mm, as well as the use of histopathological biomarkers on Senegal sole larvae for ecotoxicological testing. Eight-day old larvae of Solea senegalensis obtained from breeders' natural spawns in cultivation conditions were submitted to histological processing by an adjusted protocol comprising fixation, agarose array pre-embedding, paraffin embedding, slides preparation, and image analysis by light optical microscope coupled to a digital camera. Low-cost and easy-to-use alternatives were applied in the technique execution. The histological steps were detailed and the multiple tissues and cellular structures of the S. senegalensis larvae were described and discussed. This can be considered a valuable background information for ecotoxicological studies where changes in cell morphology and physiology are expected.