Microinjection based zebrafish embryo test for the detection of estrogenic substances in slurry based irrigation water and its combined application with yeast estrogen screen

Waste from livestock farms, including manure, is a significant source of estrogenic pollutants in the environment. These wastes have complex matrices, necessitating the implementation of in vivo and in vitro tests in order to investigate their estrogenic effects. However, most current in vivo methods are limited by the toxic effect of livestock waste due to their high concentrations of organic matter. Here we propose a novel in vivo microinjection method which is able to avoid this limitation. In this study, the estrogen content of slurry-based irrigation water extracts from dairy cattle farms was examined using a classical in vitro and the newly developed in vivo method. The limitations of the in vitro system, with its absence of endogenous steroid hormone receptors and subsequent lack of elucidating complex interactions involving the estrogen receptor (ER), are complemented by the in vivo fish test, which allows for a more complete assessment of estrogenicity and toxicity to vertebrate animals. In vitro screenings were performed with the ISO 19040–1:2018 Yeast Estrogen Screen (YES). The YES test showed estrogenic activity in all 32 tested samples, which ranged from 5 to 50518 ng/L in EEQ (E2-Estradiol equivalents). The in vivo microinjection method was developed using a Tg(vtg1:mCherry) transgenic zebrafish embryo model. This model is able to eliminate secondary symptoms of hypoxia that may occur during normal aqueous exposure to high organic matter extracts. Using the microinjection method, a total of 12 samples, out of the 32 samples examined, presented no observable estrogenic effects in fish embryos based on integrated density values. In samples where the fish test showed no estrogenic effect, the liver of the larvae was significantly damaged due to sample toxicity. Our results clearly show that the combination of these methods provides a highly effective screening tool for samples containing high concentrations of organic matter.