Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on industrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method. Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section （influents） and 9 from post-treatment section （effluents）, were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant. Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition. By comparison with a standard curve for 17β-estradiol （E2）, estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples. Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

Full sexual maturity-cycle exposure to environmentally relevant concentrations of 17β-estradiol decreases reproductive capacity of zebrafish

17β-Estradiol(E2)has been widely detected in natural water and treatment with E2 induces potential endocrine disrupting effects in fish.However,effects on fish fecundity and steroid system after treatment with environmentally relevant concentrations of E2 for the full sexual maturation cycle remain unclear.In this study,zebrafish were treatment with 0,10 or100 ng/L E2 from embryo to adult stage,and effects on gonadal development and differentiation,steroid hormone levels,transcription of genes associated with the hypothalamic-pituitary-gonadal-liver(HPG)axis in adults and fertilization rate of offspring were assessed.The results showed that treatment with E2 lead to increased number of feminization in zebrafish.In females,E2 decreased cumulative amount of spawning and inhibited the maturation of oocyte.In males,E2 inhibited the maturation and motility of sperm,as well as decreased the movement speed of sperm.These adverse effects on sperm might be responsible for the reduced fertilization observed in offspring.In addition,treatment with E2 changed the levels of steroid hormones in zebrafish gonad and altered the transcriptional levels of genes associated with HPG axis,which is responsible for the regulation of germ cells maturation and gonadal development in zebrafish.Overall,these results suggested that treatment with environmentally relevant concentrations of E2 for the full sexual maturity cycle resulted in adverse effects on reproduction in zebrafish.

Assessment of estrogen disrupting potency in animal foodstuffs of China by combined biological and chemical analyses

Food has been documented as one of major routes for human exposure to environmental estrogens（EEs）, but information on the occurrence of EEs in animal foodstuffs is still scarce.This study analyzed estrogenic activity in 16 types of animal foodstuffs（n = 142） collected from four cities（Wuhan, Guangzhou, Wenzhou and Yantai） of China by combined yeast estrogen screen（YES） bioassay and chemical analysis. By bioassay, all samples＇ extracts were found to induce estrogenic activities and the bioassay-derived 17β-estradiol equivalent（EEQbio） ranged from 8.29 to 118.32 ng/g. In addition, the samples were analyzed by liquid chromatography coupled to tandem mass spectrometry for further chemical analysis. 17β-Estradiol was found in all samples in this survey at levels of 0.44 to 15.04 ng/g.All samples had 33.1% detection rate of 17α-ethinylestradiol（EE2）, and the maximum concentration was 2.80 ng/g. Bisphenol A and 4-nonylphenols were detected in 83.8% and83.1% of samples, with concentrations up to 12.56 ng/g and 35.76 ng/g, respectively.However, the concentrations of estrone, diethylstilbestrol and 4-t-octylphenol were found to be below the limit of detection. A comparison of EEQbiomeasured from the YES assay and EEQchemcalculated from chemical analysis showed good correlation（R2= 0.84）. Based on the results, the YES assay can be used as a rapid pre-screening method for monitoring the levels of estrogenic activity in large numbers of animal foodstuffs, and chemical analysis used in combination can be used for the identification of specific EEs.