# An Easy-to-Use Histological Technique for Small Biological Samples of Senegalese Sole Larvae
## 1 Introduction  
The characterization of the effects of contaminants exposure is one stage of the environmental risk assessment (ERA) framework, which establishes essential guidelines to drive decision making on relevant environmental issues to improve health quality through management, preservation and conservation of the environment [1]. Upon exposure to stressors, organisms may undergo differential biological responses at morpho-physiological functions, named biomarkers, as indicators of either exposure or effects of xenobiotic chemicals at the sub-organism or organismal level [2]. Therefore, a multi-level biomarker approach, including biochemical, behavioral, genotoxic, physiological, molecular and histopathological responses, has been widely used within ERA scenarios [3, 4]. Histology is a traditional and fundamental technique employed in biological developmental studies or pathological investigations through the morpho-functional study of tissues, providing morphocellular description of diseases prognostic and diagnostic, their implications and understanding [5, 6, 7, 8, 9, 10, 11, 12]. Defined as the study of abnormal tissue structure, the histopathology of tissues' examination from fish and other aquatic organisms may identify the cause of the abnormality and eventually death and possibly the causative agent [6, 13]. Therefore, this method is also very useful in environmental sciences andecology, where a histological approach can demonstrate the effects of exposure to xenobotic compounds and environmental contaminants [1; 2; 3; 4; 6; 7; 8]. This information along with physiological and biochemical data may provide a more complete and accurate description of the activity of a chemical agent [13]. In assessing aquatic pollution, histological analysis using fish as a model organism has been successfully applied as a medium-term response to sub-lethal stressors, especially chronic ones, at the intermediated level of biological organization, i.e., tissues and organs [3; 7; 11; 14; 15; 16; 17; 18]. Many guideline documents that include histopathology are already validated in a regulatory context by official bodies, e.g., OECD (Organization for Economic Co-operation and Development) and EPA (US Environmental Protection Agency) [19; 20]. However, the focus of research labs shifts more and more to molecular biomarkers, and histopathology has been left out of most routine analyses due to the time-consuming process compared to other ecotoxicological tools (e.g., biochemical biomarkers). In addition, the costs and needs associated with equipment and staff training may discourage the adoption of this approach. Nevertheless, understanding the adverse effects caused by environmental stressors through histopathology techniques cannot be detected or replaced by other typical ecotoxicological protocols [3; 21; 22; 23]. In most studies, whether ecotoxicological or not, the use of small organisms is preferred due to advantages such as easy handling under laboratory conditions, availability of many organisms, reduction in space requirement, and the waste amount produced. The histological study of small organisms is not usually considered since handling small samples during processing is challenging. Thus, very few studies have described methods to perform histology of other fish larvae species, apart from the model zebrafish species (_Danio rerio_) [5; 24; 25]. For instance, the small size of zebrafish larvae and the ethical requirements for vertebrates make them particularly well suited for toxicity testing as human disease model organisms [5; 24; 25; 26]. On the other hand, the same efforts are missing for other fish larvae species of ecological relevance, such as the case of Senegelese sole, necessary for an ecosystem-scale knowledge of the marine environment. The flatfish Senegelese sole, _Soleen senegalensis_ (Kaup 1858; order Pleuronectiformes), occurs along the Europe and African Atlantic coasts and the Occidental Mediterranean is extensively exploited for aquaculture in southern European countries [27; 28; 29].