Evaluation of developmental toxicity, immunotoxicity and Endocrine Disruption Caused by Exposure to Triazines, Triazoles and Short-Chain PFAS Triazole fungicides, triazines and short-chain PFAS are generally persistent and mobile (PM) chemicals. Their high water solubility and slow degradation makes them particularly likely to pollute potable water, leading to potentially high degrees of exposure in humans. To gain more insight in the hazards of these compounds, 16 triazoles, 9 triazines and 10 PFAS were tested for developmental toxicity on wild type zebrafish (Danio rerio) embryos and for endocrine disruption on cell lines. We are still working on two immunotoxicity assays to show effects of chemical exposure on bacterial clearance and phagocyte migration in zebrafish embryos. The developmental toxicity was measured using a growth scoring system [1]. The zebrafish embryos were also checked on teratogenicity and heart rate. Benchmark doses (BMD5) for a 5% decreased response were calculated to compare the potency of the tested chemicals. The endocrine disruption was tested with reporter gene cell lines regarding hormone receptor agonism and antagonism. Also, effects on steroidogenesis were tested using a human adrenocortical (H295R) cell line and liquid chromatography tandem mass spectrometry on the supernatants Peter Cenijn Vrije Universiteit Amsterdam Majorie van Duursen Vrije Universiteit Amsterdam Wilbert Bitter Vrije Universiteit Amsterdam Introduction and methods • Nine of the tested triazole fungicides showed activity on zebrafish embryo growth and heartbeat (table 1). Four of these remain to be tested in dilution series. • The underlying mechanism has been described earlier as the inhibition of Cytochrome P450 (CYP) 26 that regulates the catabolism of retinoic acid which is an important signaling molecule during embryogenesis and cardiogenesis [2]. • These same triazole fungicides showed effects on steroid hormone synthesis (figure 2). The steroidogenic profile after exposure of most triazoles shows an increase of progesterone and 17aOH progesterone and decrease of all other steroids. This is caused by inhibition of CYP 17 and CYP 21 which prevents the transformation of these progestogens to the other hormones further down the steroidogenic cascade. • In addition to inhibiting CYP enzymes, the triazole fungicides also show antagonism towards the estrogen (ER) and androgen receptor (AR). This means that exposure to these chemicals have multiple mechanisms to exert endocrine disruption. This antagonistic activity may in turn lead to decreased expression of CYP enzymes. • In conclusion, possible pollution of potable water by triazole fungicides is a cause of concern due to their potential of endocrine disruption and embryotoxicity. Results, conclusion and discussion This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 101036756. Maxim Carlier m.p.carlier@vu.nl Vrije Universiteit Amsterdam Partners Timo Hamers Vrije Universiteit Amsterdam growth score Heartbeat ametryn BMD5: 9.815 μM no effect bitertanol BMD5: 8.811 μM BMD5: 2.802 μM fenbuconazole BMD5: 14.15 μM BMD5: 4.623 μM tebuconazole BMD5: 17.26 μM BMD5: 9.448 μM propiconazole BMD5: 13.4 μM BMD5: 2.989 μM triadimenol no effect BMD5: 5.788 μM tetraconazole to be tested to be tested cyproconazole to be tested to be tested difenoconazole to be tested to be tested paclobutrazol to be tested to be tested Table 1: BMDs for the growth scores and change in heartbeat of embryos exposed to active compounds. Prior to these dose response experiments, all 36 chemicals were tested at 100 μM. Compounds that showed toxicity were tested in serial dilutions. References: [1] Hermsen, S. A., van den Brandhof, E. J., van der Ven, L. T., & Piersma, A. H. (2011). Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies. Toxicology