Glucocorticoids in aquatic systems originating from natural excretion and medical use may pose a risk to fish. Here, we analyzed physiological and transcriptional effects of clobetasol propionate (CLO), cortisol and cortisone in zebrafish embryos as single compounds and binary mixtures. CLO and cortisol, but not cortisone showed a concentration-dependent decrease in muscle contraction, increase in heart rate, and accelerated hatching. CLO also induced immobilization and edema at high concentrations. Transcription analysis covering up to 26 genes showed that mostly genes related to glucose metabolism, immune system and development were differentially expressed at 91 ng/L and higher. CLO showed stronger effects on immune system genes than cortisol, which was characterized by upregulation of fkbp5, irg1l, gilz, and socs3, and development genes, matrix metalloproteinases mmp-9 and mmp-13, while cortisol led to stronger upregulation of the gluconeogenesis genes g6pca and pepck1. CLO also induced genes regulating the circadian rhythm, nr1d1 and per1a. In contrast, cortisone led to down-regulation of vitellogenin. Binary mixtures of cortisol and CLO mostly showed a similar activity as CLO alone on physiological and transcriptional end points but additive effects in heart rate and pepck1 upregulation, which indicates that mixtures of glucocorticoids may be of concern for developing fish.

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Fishes are exposed to mixtures of different classes of steroids, but ecotoxicological implications are not sufficiently known. Here, we systematically analyze effects of different combinations of steroid mixtures in zebrafish embryos to assess their joint activities on physiology and transcriptional alterations of steroid-specific target genes at 96 and 120 h post fertilization. In binary mixtures of clobetasol propionate (CLO) with estradiol (E2) or androstenedione (A4), each steroid exhibited its own expression profile. This was also the case in mixtures of 5-, 8-, and 13-different classes of steroids in exposure concentrations of 10–10,000 ng/L. The transcriptional expression of most genes in different mixtures was steroid-specific except for genes encoding aromatase (cyp19b), sulfotransferase (sult2st3), and cyp2k22 that were induced by androgens, progestins, and glucocorticoids. Marked alterations occurred for sult2st3 in binary mixtures of CLO + E2 and CLO + A4. Glucocorticoids increased the heart rate and muscle contractions. In mixtures containing estrogens, induction of the cyp19b transcript occurred at 10 ng/L and protc from the anticoagulation system at 100 ng/L. Our study demonstrates that steroids can act independently in mixtures; the sum of individual steroid profiles is expressed. However, some genes, including cyp19b, sult2st3, and cyp2k22, are regulated by several steroids. This joint effect on different pathways may be of concern for fish development.

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