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.

Background. Analyses of the control region sequences of European brown trout populations’ mitrochondrial DNA have revealed five main evolutionary lineages (Atlantic, Danubian, Mediterranean, Adriatic, Marble) mostly relating to the main water basins; however, the hybridization between lineages were increasingly reported. Due to the hydrogeography of Hungary, wild populations should theoretically belong to the Danubian lineage, however, this has not been verified by genetic studies. Methods. In our study multiple molecular marker sets (mitochondrial sequence, microsatellites, PCR-RFLP of nuclear markers and sex marker) were used to investigate the genetic composition and population genetics of the brown trout populations in two broodstocks, six wild streams in Hungary and one Serbian population. Results. The admixture of Atlantic and Danubian lineages in these populations, except the Serbian population with pure Danubian origin, was observed by control region sequences of mitochondrial DNA and PCR-RFLP markers in the nuclear genome, and one unpublished Danubian haplotype was found in Hungarian populations. A sex-specific marker revealed equal gender ratio in broodstocks and Kemence stream, whereas in other wild streams the proportion of female individuals were less than 50%. Structure and principal component analyses based on the alleles of microsatellite loci also revealed overlapping populations, however the populations were still significantly different from each other and were mostly in Hardy-Weinberg equilibrium. Discussion. Stocking and migration can have a significant genetic impact on trout populations of wild streams, however there are no guidelines or common practices for stocking of small streams in Hungary, thus the genetic background of these populations should be considered when developing conservation actions.