Abstract
Rainbow trout (Oncorhynchus mykiss) are frequently used as experimental animals in ecotoxicological studies, in which they are experimentally exposed to defined concentrations of test substances, such as heavy metals, pesticides, or pharmaceuticals. Following exposure to a broad variety of aquatic pollutants, early morphologically detectable toxic effects often manifest in alterations of the gills. Suitable methods for an accurate and unbiased quantitative characterization of the type and the extent of morphological gill alterations are therefore essential prerequisites for recognition, objective evaluation and comparison of the severity of gill lesions. The aim of the present guidelines is to provide practicable, standardized and detailed protocols for the application of unbiased quantitative stereological analyses of relevant morphological parameters of the gills of rainbow trout. These gill parameters inter alia include the total volume of the primary and secondary gill lamellae, the surface area of the secondary gill lamellae epithelium (i.e., the respiratory surface) and the thickness of the diffusion barrier. The featured protocols are adapted to fish of frequently used body size classes (300–2000 g). They include well-established, conventional sampling methods, probes and test systems for unbiased quantitative stereological analyses of light- and electron microscopic 2-D gill sections, as well as the application of modern 3-D light sheet fluorescence microscopy (LSFM) of optically cleared gill samples as an innovative, fast and efficient quantitative morphological analysis approach. The methods shown here provide a basis for standardized and representative state-of-the-art quantitative morphological analyses of trout gills, ensuring the unbiasedness and reproducibility, as well as the intra- and inter-study comparability of analyses results. Their broad implementation will therefore significantly contribute to the reliable identification of no observed effect concentration (NOEC) limits in ecotoxicological studies and, moreover, to limit the number of experimental animals by reduction of unnecessary repetition of experiments.
Highlights
In ecotoxicological studies, the rainbow trout (O. mykiss) is frequently used as a sensitive experimental fish species to examine the effects of various surface water pollutants, including diverse chemicals, pharmaceuticals, heavy metals, as well as solid particles such as microplastic, on aquatic organisms [1,2,3,4,5]
This is especially relevant for the evaluation of histopathological alterations in experimental animals exposed to low concentrations of test substances, where lesions might be subtle and not manifested in all individuals, respectively in all examined samples of one animal [7,8,14]
The broad implementation of the featured methods will significantly contribute to the representativity, unbiasedness, reliability and comparability of analyses results in ecotoxicology studies reporting quantitative morphological gill parameters and add urgently required certainty to the detection of no observed effect concentration (NOEC) values as a base for the specification of legal concentration limits of aquatic pollutants
Summary
The rainbow trout (O. mykiss) is frequently used as a sensitive experimental fish species to examine the effects of various surface water pollutants, including diverse chemicals, pharmaceuticals, heavy metals, as well as solid particles such as microplastic, on aquatic organisms [1,2,3,4,5]. Quantitative stereology of the gills of rainbow trout in ecotoxicological studies In the outlined experimental approach (Section 4), the analysis of histological sections yields relative quantities of the examined morphological parameter per volume unit of the reference compartment (i.e., volume-, surface area-, length densities and numerical volume densities). Interpretation of these relative parameters alone, may be inconclusive, since changed densities may result from changes of the target structures, as well as from altered volumes of the reference compartments, or both, which is referred to as the “reference trap” [25,67]. For a more detailed discussion of the general principles of quantitative stereology, the interested reader is referred to the standard textbooks of stereology and several excellent reviews of quantitative stereological analyses in biomedical research [25,26,32,71]
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