Avaliação dos fatores de interferência no ensaio in vitro Yeast Estrogen Screen (YES) para análise de estrogenicidade em amostras ambientais, compostos orgânicos e misturas

Abstract

Bioassays have been used for the monitoring of endocrine disruptors in the environment. For this reason, the interest in low quantification and detection limits has increased. The Yeast Estrogen Screen – YES assay identifies the xenoestrogens effects through the transcriptional activation of ER, providing information on total estrogenic activity in bioequivalence (17β-estradiol equivalent – E2-EQ). The aim of this work was to evaluate the possible interferences on this assay, some of them were analyzed, such as variation of initial cellular density, incubation duration, wavelength used for the reading and vehicle of the test reference substance of the YES assay. Environmental samples (drinking water, surface water, effluent and affluent from sewage treatment plant, raw leachate and treated leachate and sediment), organic compounds (17β-estradiol, 17α-ethinyl estradiol, estrone, estriol, bisphenol-A, ethylparaben, butylparaben, methylparaben, propylparaben, propylparaben, propylparaben, atrazine, simazine, triclosan, caffeine and nonylphenol) and mixtures of estrogens (17β-estradiol, 17α-ethinylestradiol, estrone, estriol) and bisphenol-A were selected as evaluating tools on this work. The initial cell density and the incubation time length of the assay influenced directly on the method sensitivity. However, the results expressed in E2-EQ and RP did not show any variation. Surface water showed similar responses for the 48 hours and 72 hours of incubation. In contrast, EC50 results showed dependency on these factors and the usage of control-chart has shown more reliable results since it allows to monitor the average of EC50 and its dispersion. The lowest limit of detection calculated for the method was 7 ± 3 ng L-1 and reached 0.28 ± 0.12 ng L-1 after the division by the highest final concentration factor applied to the environmental matrix. Likewise, the lowest limit of quantification calculated was 21 ± 10 ng L-1 reaching 0.84 ± 0.4 ng L-1 after considering the sample final concentration factor. These limits meet the maximum LOD proposed by document 2018/840/EU for E2 monitoring in surface waters by chromatographic methods. In addition, assessment of interfering factors in YES assay towards evaluation of estrogenicity in environmental samples, organic compounds and chemical mixtures provided an optimized protocol and a list of recommendations for the implementation and performance of the YES assay with safe and reliable results.