Bioluminescent Yeast Estrogen Screen Research Articles

Identification, Elucidation, and Toxicity Assessment of Nontarget Disinfection By-products from Fipronil Chlorination

In the present study, fipronil degradation was evaluated in typical chlorination conditions (fipronil: 20 μg L−1; free chlorine concentration: 2 mg L−1; pH 7.5; 25 ○C). Fipronil was quickly degraded by chlorination with half-time life of 2.08 min, achieving 95% of degradation in 15 min of reaction. For identification and elucidation of its disinfection by-products, fipronil solutions (2.0 mg L−1) were chlorinated at different contact times. Six by-products were identified and their structural formulas were elucidated by high-resolution mass spectrometry. Lastly, the toxicity of the unreacted and chlorinated solutions was evaluated using in vitro, in vivo, and in silico assays. Unreacted and chlorinated solutions were toxic to Artemia salina nauplii with an increase on toxic effects after 24 h of reaction time. No estrogenicity and cytotoxicity were observed for the tested solutions using re-engineered bioluminescent yeast estrogen screen and colorimetric MTT assays, respectively. According to quantitative structure-activity relationship methodology, chlorination may produce a more mutagenic compound than the precursor fipronil. The generated disinfection by-products also proved to be developmental toxicant, although it was not possible to measure their relative potency in relation to the parent compound.

Aqueous chlorination of herbicide metribuzin: Identification and elucidation of “new” disinfection by-products, degradation pathway and toxicity evaluation

A widely used herbicide, metribuzin, was evaluated for degradation, mineralization and disinfection by-products (DBPs) formation during aqueous chlorination. In addition, to assess the toxicity effects of chlorination on metribuzin solution the following tests were performed: acute toxicity using Artemia salina nauplii; cell viability using MTT assay; estrogenicity using a re-engineered Bioluminescent Yeast Estrogen Screen (BLYES) and a constitutively bioluminescent strain (BLYR); mutagenicity and developmental toxicity using Q(SAR) methodology. Metribuzin at 10 mg·L−1 was degraded by chlorination, achieving 93% of removal at 30 min of reaction. TOC analysis showed that the herbicide does not suffer complete mineralization, even after 24 h of contact with free chlorine. Seventeen DBPs were detected and their structural formulae were elucidated by high resolution mass spectrometry. Toxicity effects for chlorinated solutions increased when compared to the unreacted metribuzin solution. DBPs were more toxic to Artemia salina nauplii, increasing around 20% on nauplii mortality. It was also observed high estrogenicity to human receptors in BLYES assays and mutagenic and developmental toxicant effects to animals and humans in Q(SAR) methodology, suggesting that DBPs are potentially more toxic than the precursor metribuzin. Metribuzin solutions at 10 mg·L−1 showed equivalent 17-β-estradiol values ranged from 0.061 to 6.71 µg·L−1 after to be chlorinated at different reaction times.

Assessing the Estrogenic Activity of EDCs and Human Risks of Groundwater after Ozonation and Chlorination

ABSTRACTEndocrine Disrupting Chemicals (EDCs) in groundwater could result in severe risks to human health. Therefore, in the present study, ozone (O3) and chlorine were applied to remove the estrogenicity of Bisphenol-A (BPA), 4-Nonylphenol (4-NP) and Triclosan (TCS) in groundwater. Bioluminescent Yeast Estrogen Screen and Gas Chromatography Mass Spectrometry (BLYES-GC/MS) demonstrated that there is no directly proportional relationship between concentration and estrogenicity. Human Risk Assessment (HRA) was conducted, considering the concentration of EDCs, their estrogenicity and the by-products formed. Estrogenicity decreased by 98.7, 79.3 and 97.0% (ozone) and 86.2, 94.3 and 97.8% (chlorine) for BPA, 4-NP AND TCS, respectively. The individual Hazard Quotient (HQ<1), cumulative risk Hazard Index (HI) and intake 17-estradiol equivalents (EEQ) (µg day−1) indicate that ozone and chlorine treatment, including any by-products formed, do not result in estrogenicity with associated HR.

Estrogen sensitive liver transgenic zebrafish (Danio rerio) line (Tg(vtg1:mCherry)) suitable for the direct detection of estrogenicity in environmental samples

Environmental estrogens are a serious concern worldwide due to their ubiquity and adverse ecotoxicological and health effects. Chemical structure of these substances is highly diverse, therefore estrogenicity cannot be predicted on the basis of molecular structure. Furthermore, estimation of estrogenicity of environmental samples based on chemical analytics of suspects is difficult given the complex interaction of chemicals and the impact on estrogenicity. The full estrogenic impact of an environmental sample can thus only be revealed by a series of sensitive in vitro and in vivo ecotoxicological tests. Herein we describe a vitellogenin reporter transgenic zebrafish line (Tg(vtg1:mCherry)) that enables the detection of estrogenicity in the environmentally relevant, low concentration ranges in embryonic tests that are in accordance with 3Rs and relevant animal welfare regulations.The transgene construct used for the development of Tg(vtg1:mCherry) carried a long (3.4 kbp) natural vitellogenin-1 promoter sequence with a high number of ERE sites.A test protocol was developed based on our finding that the endogenous vitellogenin and the reporter show similar spatial expression pattern and both endogenous and vitellogenin reporter is only produced in the left hepatic lobe of 5 dpf zebrafish embryos.Seven generations of Tg(vtg1:mCherry) have been established, and the estrogen responsiveness was tested with different estrogenic substances and wastewater samples. Embryos were exposed from 3 to 5 days post fertilization (dpf). Fluorescence in embryos could be detected upon treatment with 17-ß-estradiol from a concentration of 100 ng/L, 17-α-ethynilestradiol from 1 ng/L, zearalenone from 100 ng/L and bisphenol-A from 1 mg/L. In the adult stage transgene activity appeared to be more sensitive to estrogen treatment, with detectable transgene activity from 5 ng/L 17-ß-estradiol concentration. The transgenic line Tg(vtg1:mCherry) was also suitable for the direct measurement of estrogenicity in wastewater samples without sample extraction. The detection of estrogenic activity using the reporter line was confirmed by the bioluminescent yeast estrogen screen.

A novel bioluminescent NanoLuc yeast-estrogen screen biosensor (nanoYES) with a compact wireless camera for effect-based detection of endocrine-disrupting chemicals.

The presence of chemicals with estrogenic activity in surface, groundwater, and drinking water poses serious concerns for potential threats to human health and aquatic life. At present, no sensitive portable devices are available for the rapid monitoring of such contamination. Here, we propose a cell-based mobile platform that exploits a newly developed bioluminescent yeast-estrogen screen (nanoYES) and a low-cost compact camera as light detector. Saccharomyces cerevisiae cells were genetically engineered with a yeast codon-optimized variant of NanoLuc luciferase (yNLucP) under the regulation of human estrogen receptor α activation. Ready-to-use 3D-printed cartridges with immobilized cells were prepared by optimizing a new procedure that enables to produce alginate slices with good reproducibility. A portable device was obtained exploiting a compact camera and wireless connectivity enabling a rapid and quantitative evaluation (1-h incubation at room temperature) of total estrogenic activity in small sample volumes (50μL) with a LOD of 0.08nM for 17β-estradiol. The developed portable analytical platform was applied for the evaluation of water samples spiked with different chemicals known to have estrogen-like activity. Thanks to the high sensitivity of the newly developed yeast biosensor and the possibility to wireless connect the camera with any smartphone model, the developed configuration is more versatile than previously reported smartphone-based devices, and could find application for on-site analysis of endocrine disruptors. Graphical abstract Wireless effect-based detection of endocrine-disrupting chemicals with nanoYES platform.

In Vitro and In Vivo Assessment of Aqueously Extractable Estrogens in Poultry Manure after Pilot-scale Composting.

Poultry manure contains free and conjugated forms of the natural estrogens 17β-estradiol and estrone, which can be transported to receiving waters via runoff when land-applied. Previous studies have demonstrated estrogens in runoff from poultry manure-amended fields but have not tracked changes in estrogenicity within this water over time. Microbial conversion of conjugated estrogens (a major portion of water-extractable estrogens) to parent forms may result in temporary increases in estrogenicity in natural water bodies. The present study created 80-L batches of simulated poultry manure runoff, which were investigated over 10 d for estrogenicity by bioluminescent yeast estrogen screen assay and fathead minnow () vitellogenin induction model. The efficacy of different compost conditions (in-vessel aeration ± turning, and piling) on reduction/elimination of aqueously extractable estrogens in poultry manure was also investigated. Results indicate 3- to 10-fold increases in estrogenicity in various poultry manure mixtures during 10-d observations. Estrogenicity returned to low levels in postcompost treatments but remained elevated in the precompost treatment. Aerated compost resulted in >75% reductions in initial, peak, and 10-d mean estrogenicity in aqueous mixtures (0.3, 0.8, and 0.5 ng 17β-estradiol equivalents [EEQ] L, respectively) compared with the precompost mixture (1.4, 4.8, and 2.1 ng EEQ L, respectively). Estrogenicity was significantly higher in the aqueous extract from the piled treatment than the aerated treatment, and 10-d exposure of male fish to the piled treatment resulted in statistically significant vitellogenin induction. Collectively, our results suggest a need to investigate estrogenicity in surface waters for several days after receiving manure-influenced runoff.

Investigação da atividade estrogênica em águas subterrâneas do Estado de São Paulo

Os interferentes endócrinos são substâncias químicas de diversas naturezas como hormônios naturais (estrona - E1, estradiol - E2, estriol - E3), hormônios sintéticos (etinilestradiol - EE2, utilizado em anticoncepcionais), compostos orgânicos sintéticos como o bisfenol A, praguicidas organoclorados como o DDT, entre outros, capazes de interferir na ação dos hormônios. Com o atual quadro de demanda hídrica no Estado de São Paulo e consequente aumento da exploração de água subterrânea, o potencial de infiltração de água superficial contaminada por interferentes endócrinos na água subterrânea tem aumentado devido ao rebaixamento do lençol, especialmente nos locais onde poços de alta vazão estão localizados próximos às águas superficiais. As redes de esgotos danificadas e sistemas sépticos podem também levar a lixiviação desses compostos para as águas subterrâneas. Com objetivo de investigar a atividade estrogênica em águas subterrâneas do Estado de São Paulo, a CETESB utilizou no presente estudo o bioensaio BLYES (Bioluminescent Yeast Estrogen Screen), baseado em leveduras bioluminescentes, no período de 2011 a 2015. Dos 78 pontos de coleta da rede de monitoramento, 6 apresentaram atividade estrogênica superior ao limite de quantificação do método, entretanto, os valores ficaram abaixo do valor de referência de 3,8 ng E2 eq L-1, evidenciando que não há risco à saúde humana para consumo de água subterrânea a longo prazo. Os resultados mostram que as águas subterrâneas analisadas estão protegidas quanto à ocorrência de atividade estrogênica.

Integrated Assessment of Aqueously Extractable Estrogens in Municipal Biosolids after Pilot-Scale Composting

Abstract. Municipal biosolids contain natural estrogens, e.g., 17ß-estradiol (E2) and estrone (E1), and synthetic estrogens, e.g., 17a-ethinylestradiol (EE2), which can be transported to receiving waters via runoff when land-applied as fertilizer. Previous studies have investigated estrogens in runoff from biosolids-amended fields but have not tracked changes in estrogenicity within this water over time. Microbial conversion of conjugated estrogens (a major portion of water-extractable estrogens) to parent forms may result in temporary increases in estrogenicity in natural water bodies. The present study measured aqueously extractable estrogens in simulated runoff generated using biosolids sourced from four municipal wastewater treatment facilities in Maryland. Estrogen analytes (E2 and E1) were quantified over 10 d periods in 80 L static simulated runoff mixtures (0.5 g biosolids L-1) by LC-MS/MS, and estrogenic activity was estimated using the bioluminescent yeast estrogen screen (BLYES) assay and the fathead minnow () vitellogenin (VTG) induction model. The effectiveness of pilot-scale in-vessel, aerated, turned (IVAT) composting to reduce or eliminate aqueously extractable estrogens in biosolids was also investigated. On arrival, only one of four biosolids had environmentally meaningful levels of aqueously extractable estrogens. Results indicate that estrogens/estrogenicity increased &amp;gt;6.5-fold during the first 6 d in an aqueous extract generated with that sample, as indicated both by BLYES quantitation of estrogenicity (0.8 to 5.6 ng EEQ L-1) and LC-MS/MS quantitation of estrone (13.8 to 93.0 ng L-1). Results also indicate that IVAT composting was effective at reducing or eliminating aqueously extractible estrogens in that biosolids sample. This study demonstrates the need for low-level detection of estrogens/estrogenicity in complex environmental media and validates the benefits of screening assays and fish models as complementary sensitive tools for environmental monitoring. Keywords: Biosolids, Compost, Estrogenicity, Fathead minnow, Microbial deconjugation, Vitellogenin.

Investigating the influence of nitrate nitrogen on post-smolt Atlantic salmon Salmo salar reproductive physiology in freshwater recirculation aquaculture systems

A major issue affecting land-based, closed containment Atlantic salmon Salmo salar growout production in water recirculation aquaculture systems (RAS) is precocious male maturation, which can negatively impact factors such as feed conversion, fillet yield, and product quality. Along with other water quality parameters, elevated nitrate nitrogen (NO3-N) has been shown to influence the reproductive development and endogenous sex steroid production in a number of aquatic animal species, including Atlantic salmon. We sought to determine whether elevated NO3-N in RAS can influence early maturation in post-smolt Atlantic salmon in an 8-month trial in replicated freshwater RAS. Post-smolt Atlantic salmon (102±1g) were stocked into six RAS, with three RAS randomly selected for dosing with high NO3-N (99±1mg/L) and three RAS set for low NO3-N (10±0mg/L). At 2-, 4-, 6-, and 8-months post-stocking, 5 fish were randomly sampled from each RAS, gonadosomatic index (GSI) data were collected, and plasma was sampled for 11-ketotestosterone (11-KT) quantification. At 4- and 8-months post-stocking, samples of culture tank and spring water (used as “makeup” or replacement water) were collected and tested for a suite of 42 hormonally active compounds using liquid chromatography/mass spectrometry, as well as for estrogenicity using the bioluminescent yeast estrogen screen (BLYES) reporter system. Finally, at 8-months post-stocking 8–9 salmon were sampled from each RAS for blood gas and chemistry analyses, and multiple organ tissues were sampled for histopathology evaluation. Overall, sexually mature males were highly prevalent in both NO3-N treatment groups by study’s end, and there did not appear to be an effect of NO3-N on male maturation prevalence based on grilse identification, GSI, and 11-KT results, indicating that other culture parameters likely instigated early maturation. No important differences were noted between treatment groups for whole blood gas and chemistry parameters, and no significant tissue changes were noted on histopathology. No hormones, hormone conjugates, or mycotoxins were detected in any water samples; phytoestrogens were generally detected at low levels but were unrelated to NO3-N treatment. Finally, low-level estrogenicity was detected in RAS water, but a NO3-N treatment effect could not be determined. The major findings of this study are i) the NO3-N treatments did not appear to be related to the observed male maturation, and ii) the majority of hormonally active compounds were not detectable in RAS water.

Effects of watershed densities of animal feeding operations on nutrient concentrations and estrogenic activity in agricultural streams

Application of manures from animal feeding operations (AFOs) as fertilizer on agricultural land can introduce nutrients and hormones (e.g. estrogens) to streams. A landscape-scale study was conducted in the Shenandoah River watershed (Virginia, USA) in order to assess the relationship between densities of AFOs in watersheds of agricultural streams and in-stream nutrient concentrations and estrogenic activity. The effect of wastewater treatment plants (WWTPs) on nutrients and estrogenic activity was also evaluated. During periods of high and low flow, dissolved inorganic nitrogen (DIN) and orthophosphate (PO4-P) concentrations were analyzed and estrogens/estrogenic compounds were extracted and quantified as17β-estradiol equivalents (E2Eq) using a bioluminescent yeast estrogen screen. Estrogenic activity was measurable in the majority of collected samples, and 20% had E2Eq concentrations >1ng/L. Relatively high concentrations of DIN (>1000μg/L) were also frequently detected. During all sampling periods, there were strong relationships between watershed densities of AFOs and in-stream concentrations of DIN (R2=0.56–0.81) and E2Eq (R2=0.39–0.75). Relationships between watershed densities of AFOs and PO4-P were weaker, but were also significant (R2=0.27–0.57). When combined with the effect of watershed AFO density, streams receiving WWTP effluent had higher concentrations of PO4-P than streams without WWTP discharges, and PO4-P was the only analyte with a consistent relationship to WWTPs. The results of this study suggest that as the watershed density of AFOs increases, there is a proportional increase in the potential for nonpoint source pollution of agricultural streams and their receiving waters by nutrients, particularly DIN, and compounds that can cause endocrine disruption in aquatic organisms.

REPRODUCTIVE HEALTH OF BASS IN THE POTOMAC, USA, DRAINAGE: PART 2. SEASONAL OCCURRENCE OF PERSISTENT AND EMERGING ORGANIC CONTAMINANTS

The seasonal occurrence of organic contaminants, many of which are potential endocrine disruptors, entering the Potomac River, USA, watershed was investigated using a two-pronged approach during the fall of 2005 and spring of 2006. Passive samplers (semipermeable membrane device and polar organic chemical integrative sampler [POCIS]) were deployed in tandem at sites above and below wastewater treatment plant discharges within the watershed. Analysis of the samplers resulted in detection of 84 of 138 targeted chemicals. The agricultural pesticides atrazine and metolachlor had the greatest seasonal changes in water concentrations, with a 3.1- to 91-fold increase in the spring compared with the level in the previous fall. Coinciding with the elevated concentrations of atrazine in the spring were increasing concentrations of the atrazine degradation products desethylatrazine and desisopropylatrazine in the fall following spring and summer application of the parent compound. Other targeted chemicals (organochlorine pesticides, polycyclic aromatic hydrocarbons, and organic wastewater chemicals) did not indicate seasonal changes in occurrence or concentration; however, the overall concentrations and number of chemicals present were greater at the sites downstream of wastewater treatment plant discharges. Several fragrances and flame retardants were identified in these downstream sites, which are characteristic of wastewater effluent and human activities. The bioluminescent yeast estrogen screen in vitro assay of the POCIS extracts indicated the presence of chemicals that were capable of producing an estrogenic response at all sampling sites.