Detection of Diclofenac-Induced Alterations in Rainbow Trout (Oncorhynchus mykiss) Using Quantitative Stereological Methods

Abstract

In 2013, the nonsteroidal anti-inflammatory drug diclofenac (DCF) was included in the watch list for emerging pollutants under the European Union Water Framework Directive. Frequently, monitoring data revealed DCF concentrations in surface waters exceeding the proposed environmental quality standards of 0.04 µg L−1 and 0.126 µg L−1. In recent literature, the possible effects of DCF on fish are discussed controversially. To contribute to a realistic risk assessment of DCF, a 28-day exposure experiment was carried on rainbow trout (Oncorhynchus mykiss). To warrant reliability of data, experiments were conducted considering the Criteria for Reporting and Evaluating Ecotoxicity Data. The test concentrations of DCF used (0.1, 0.5, 1, 5, 25, and 100 µg L−1) also included environmentally relevant concentrations. The lowest-observed-effect concentration (LOEC) for a significant decrease in the plasma concentrations of the DCF biomarker prostaglandin E2 was 0.5 µg L−1 (male fish). For objective evaluation of relevant histomorphological parameters of gills and trunk kidneys, unbiased quantitative stereological methods were applied. In the gills, significant increases in the thickness of the secondary lamella and in the true harmonic mean of barrier thickness in secondary lamellae were present at DCF concentrations of 25 µg L−1 and 100 µg L−1. In the trunk kidneys, the absolute and relative volumes of nephrons were significantly decreased, paralleled by a significant increase of the volume of the interstitial renal tissue. With regard to quantitative histomorphological alterations in the trunk kidney, the observed LOEC was 0.5 µg L−1. The quantitative histomorphological analyses that were conducted allow identification and objective quantification of even subtle but significant morphological effects and thus provide an important contribution for the comparability of study results for the determination of no-observed-effect concentrations (NOEC). Environ Toxicol Chem 2023;42:859–872. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.