Interlaboratory comparison of in vitro bioassays for screening of endocrine active chemicals in recycled water

Abstract

In vitro bioassays have shown promise as water quality monitoring tools. In this study, four commercially available in vitro bioassays (GeneBLAzer(®) androgen receptor (AR), estrogen receptor-alpha (ER), glucocorticoid receptor (GR) and progesterone receptor (PR) assays) were adapted to screen for endocrine active chemicals in samples from two recycled water plants. The standardized protocols were used in an interlaboratory comparison exercise to evaluate the reproducibility of in vitro bioassay results. Key performance criteria were successfully achieved, including low background response, standardized calibration parameters and high intra-laboratory precision. Only two datasets were excluded due to poor calibration performance. Good interlaboratory reproducibility was observed for GR bioassay, with 16-26% variability among the laboratories. ER and PR bioactivity was measured near the bioassay limit of detection and showed more variability (21-54%), although interlaboratory agreement remained comparable to that of conventional analytical methods. AR bioassay showed no activity for any of the samples analyzed. Our results indicate that ER, GR and PR, were capable of screening for different water quality, i.e., the highest bioactivity was observed in the plant influent, which also contained the highest concentrations of endocrine active chemicals measured by LC-MS/MS. After advanced treatment (e.g., reverse osmosis), bioactivity and target chemical concentrations were both below limits of detection. Comparison of bioassay and chemical equivalent concentrations revealed that targeted chemicals accounted for ≤5% of bioassay activity, suggesting that detection limits by LC-MS/MS for some chemicals were insufficient and/or other bioactive compounds were present in these samples. Our study demonstrated that in vitro bioassays responses were reproducible, and can provide information to complement conventional analytical methods for a more comprehensive water quality assessment.

Trophic transfer and effects of DDT in male hornyhead turbot (Pleuronichthys verticalis) from Palos Verdes Superfund site, CA (USA) and comparisons to field monitoring

“…Transactivation receptor assays have been used to assess individual chemical binding affinity in environmental matrices (Wilson et al, 2004), as well as to assess estrogenic and anti-androgenic activity in fish bile following exposure to effluent (Hill et al, 2010). Effect-based in vitro assays also have been used for surface and drinking water assessments (Escher et al, 2014, Mehinto et al, 2015). The role of in vitro effect-based assays may increase as the USEPA continues bioassay use in ToxCast High Throughput System (HTS) assessments (USEPA, 2014) and in environmental risk assessments (Doyle et al, 2014).…”

Linking in vitro estrogenicity to adverse effects in the inland silverside (Menidia beryllina)

“…The estrogenicity of wastewater effluent samples was calculated as described in Mehinto et al (2015), and expressed as bioanalytical equivalent concentration (ER-BEQ; in ng E2/L AE standard deviation). The estrogenicity of wastewater effluent samples was calculated as described in Mehinto et al (2015), and expressed as bioanalytical equivalent concentration (ER-BEQ; in ng E2/L AE standard deviation).…”

“…Cell assay data were plotted to generate a dose-response curve for each estrogenic chemical and derive 10 and 50% effect concentrations (EC10 and EC50). The estrogenicity of wastewater effluent samples was calculated as described in Mehinto et al (2015), and expressed as bioanalytical equivalent concentration (ER-BEQ; in ng E2/L AE standard deviation). Changes in relative gene expression (AE standard error of the mean) were calculated using the comparative CT method described by Schmittgen and Livak (2008).…”

“…Among the bioactive chemicals most frequently detected are those exerting estrogenic activity, with reported estradiol equivalent concentrations between 1 and 100 ng/L (Leusch et al 2010;Conley et al 2017). Studies have reported differential sensitivity of mammal-and fish-based cell lines to EDCs and cytotoxic chemicals (G€ ulden et al 2005;Wilson et al 2007), but mammalian cell lines have in particular been shown to be highly sensitive and reproducible (Leusch et al 2010;Mehinto et al 2015), suggesting their utility as a conservative screen for protection of ecological as well as human health. However, before mammalian cell assays can be used for ecotoxicity screening, it is important to characterize the linkage between their in vitro bioactivity and ecotoxicologically relevant outcomes in vivo.…”

Evaluation of genotoxic effects of surface waters using a battery of bioassays indicating different mode of action

“…Bioassays that evaluate potential genotoxic effects of source and river waters are essential (Charalampous et al, 2015;Radic et al, 2010;Warren et al, 2015;Yu et al, 2011). In vitro bioassays provide high-throughput, shortterm, and low-cost measurements of potential toxicity of chemicals and have been used frequently in water quality assessments in recent decades (Jiang et al, 2012;Mehinto et al, 2015;Wei et al, 2012). A single bioassay can detect specific effects of chemicals that have the identical endpoint or mode of action (MOA).…”