Estrogen 17α-ethinylestradiol Research Articles

Performance of tube-in-tube membrane microreactor in E2/EE2 degradation via UV/H2O2/TiO2 process

The tube-in-tube membrane microreactor is an advanced system designed for the continuous removal of contaminants of emerging concerns (CECs) from urban and industrial wastewater. Despite its high removal efficiency, critical questions remain regarding the impact of various operational parameter: (i) flow rate of contaminants, (ii) oxidant dosage, (iii) concentration of the H2O2 stock solution, and (iv) pH of the H2O2 stock solution. This study addresses these questions, with particular focus on the previously unexplored effects of H2O2 concentration and pH. We investigated the degradation of estrogens 17β-Estradiol (E2) and 17α-Ethinylestradiol (EE2) using a UV/H2O2/TiO2 process using a membrane tube-in-tube reactor assembled with an inner glass fiber microfiltration membrane, coated with the catalyst TiO2-P25, and an outer quartz tube. Synthetic contaminated solution [E2/EE2]inlet = 500μgL-1 was fed in the reactor annulus and the H2O2 stock solution with different pHs (2, 3, 4, 5, 6.8, 8) and concentrations ([H2O2]stock solution = 3000, 6000, 9000 and 12000mgL-1) was fed through the membrane pores (radial permeation) or injected upstream the reactor inlet with different dosages ([H2O2]inlet = 0, 5, 10, 15, 20mgL-1) by means of a syringe pump. Experiments were performed under UVC irradiation and dark conditions. The highest observed removal rate was, approximately, 2.9µM/min for both estrogens, achieved after 7min of reactor operation, highlighting the reactor’s potential for effective CEC degradation under optimized conditions.

Experimental exploration of estrogenic effects of norethindrone and 17α-ethinylestradiol on zebrafish (Danio rerio) gonads

Synthetic progestins and xenoestrogens found in aquatic habitats are currently gaining attention on global scale. The current study aimed to investigate the time-and dose-dependent effects of synthetic progestin Norethindrone (NET; 100, 500 and 1000 ng/L) and estrogen 17α-ethinylestradiol (EE2; 100 ng/L) individually as well as in binary mixture (1000 ng/L NET + 100 ng/L EE2) on reproductive histology and transcriptional expression profile of genes in adult zebrafish. For this, 20 female (3.15 ± 0.18 cm & 0.33 ± 0.06 g) and 20 male zebrafish in each group (2.93 ± 0.13 cm & 0.29 ± 0.04 g) were exposed to drugs dissolved in water for 30 days in 12 L rectangular tanks. We found that both NET and EE2 exposure reduced the gonadosomatic index in females, while only EE2 exposure caused significant reduction in males (p ≤ 0.05). Interestingly, NET delayed oocyte maturation in females and accelerated spermatogenesis in males, while EE2 consistently suppressed sperm maturation throughout the experiment. Further, qRT-PCR results revealed differential expression pattern of the study genes (er-α, er-β1, er-β2, pgr, vegfaa and p53) in male and female zebrafish. Co-exposure indicated potential inconsistencies in steroidal function in mixtures rather than single exposures. Our findings imply that changes in gonadal histology after NET and EE2 exposure may result from unique regulation of steroid hormone receptors. Additionally, significantly reduced p53 levels (p ≤ 0.05) following co-exposure in both sexes may suggest an elevated risk of neoplastic transformations. Further research with mammalian models will help to explore the mechanisms behind differing effects of alone and co-exposures.

Reaction thermodynamics as a constraint on piscine steroidogenesis flux distributions

While a considerable amount is known of the dynamics of piscine steroidogenesis during reproduction, the influence of thermodynamics constraints on its control has not been studied. In this manuscript, Gibbs free energy change of reactions was calculated for piscine steroidogenesis using the in silico eQuilibrator thermodynamics calculator. The analysis identified cytochrome P450 (cyp450) oxidoreductase reactions to have more negative Gibbs free energy changes relative to hydroxysteroid (HSD) and transferase reactions. In addition, a more favorable Gibbs free energy change was predicted for the Δ5 (cyp450 catalyzed) vs. Δ4 (HSD catalyzed) steroidogenesis branch-point, which converts pregnenolone to 17α-hydroxypregnenolone or progesterone respectively. Comparison of in silico predictions with in vivo experimentally measured flux across the Δ5 vs. Δ4 branch-point showed higher flux through the thermodynamically more favorable Δ5 pathway in reproducing or spawning vs. non-spawning fathead minnows (Pimephales promelas). However, the exposure of fish to endocrine stressors such as hypoxia or the synthetic estrogen 17α-ethinylestradiol (EE2), resulted in increased flux through both Δ5 and Δ4 pathways, indicating an adaptive response to increase steroidogenic redundancy. The correspondence of elevated flux through the Δ5 branch-point in spawning fish indicated the use of a thermodynamically favorable pathway to optimize steroid hormone productions during reproduction. We hypothesize that such selective use of a thermodynamically favorable steroidogenesis pathway may conserve reduced equivalents or transcriptional costs for investment to other biosynthetic or catabolic reactions to support reproduction. If generalizable, such an approach can provide novel insights into the structural principles and regulation of steroidogenesis or other metabolic pathways.

Machine learning model optimization for removal of steroid hormones from wastewater

In the past few decades, there has been a significant focus on detecting steroid hormones in aquatic environments due to their influence on the endocrine system. Most compounds of these pollutants are the natural steroidal estrogens, i.e., estrone (E1), 17β-Estradiol (E2), and the synthetic estrogen 17α-Ethinylestradiol (EE2). The Moving-Bed Biofilm Reactor (MBBR) technique is appropriate for eliminating steroid hormones. This study centers on creating a model to estimate the effectiveness of the MBBR system regarding its ability to eliminate E1, E2, and EE2. The results were modeled with artificial neural networks (ANNs). The Particle Warm Optimization (PSO) and Levenberg Marquardt (LM) algorithms were selected for network training. The models incorporated five input parameters, encompassing the COD loading rate, initial levels of E1, E2, and EE2 steroid hormones, and Hydraulic Retention Time (HRT). The optimum removal conditions (three steroid hormones and COD) were determined using the optimized ANN based on both PSO and LM algorithms. The optimal transfer functions for the hidden and output layers were identified as tan-sigmoid and linear, respectively. The best ANN structures (Neurons in input, hidden, and output layers) and correlation coefficients (R) were 5:9:4, with R = 0.9978, and 5:10:4, with R = 0.9982 for the trained networks with LM and PSO algorithms, respectively. Eventually, the input parameters' importance was ranked using sensitivity analysis (SA) through Pearson correlation and developed ANNs.

DNA damage, alterations in the expression of antioxidant enzyme genes and in the histoarchitecture of gill cells of zebrafish exposed to 17-α-ethinylestradiol

Endocrine disruptors, such as estrogen, are chemical substances with the potential to alter the hormonal balance of organisms. Their origin can be natural or artificial, and they can act at very low doses. The estrogen 17α-ethinylestradiol (EE2) is used worldwide as an oral contraceptive and is a potential contaminant in aquatic ecosystems. It is well documented that these environmental pollutants can act directly or indirectly on the reproductive system, impairing development and fertility. However, little is known about the alteration of the cell oxidative status induced by EE2. The main objective of this study was to evaluate the effect on the gill cells of adult zebrafish exposed in vivo to EE2, analyzing cell histology, DNA damage and the expression levels of genes encoding the main enzymes involved in oxidative stress pathways. The histological study showed that EE2 produces moderate to high damage to the gill tissue, an increase in gill cell DNA damage and the mRNA levels of the genes corresponding to the manganese superoxide dismutase (Mn-sod) and catalase (cat) after exposure to 5 ng/L EE2. The results indicate that EE2 causes tissue alterations, DNA damage and oxidative stress. EE2 produced important alterations in the gills, a fundamental organ for the survival of fish. There is a clear need for further research on the ecological consequences of EDCs on non-target organisms.

Peroxymonosulfate enhanced photoelectrocatalytic degradation of 17α-ethinyl estradiol

This work aimed at the degradation of the synthetic estrogen 17α-ethinyl estradiol (EE2) via a combined photoelectrocatalysis (PEC) and peroxymonosulfate (PMS) process. Pristine and combined TiO2/WO3 photoanodes were fabricated and used in this work. The removal of 1 mg L−1 of EE2 increased from 47.9 % to 88.8 % in 60 min by adding 10 mg L−1 of PMS and an external bias of 1 V. EE2 was degraded in the presence of 0.05 M sodium sulfate as the electrolyte. The rate constant for the combined PEC-PMS process was 0.044 min−1 which was higher than the rate constant of light-activated PMS process (0.0053 min−1) and PEC process (0.01 min−1). The rate of EE2 degradation was increased with an increase in the voltage and PMS concentration. EE2 removal was decreased and reached 62.5 % and 48.5 % when 0.05 M NaCl or 0.05 M NaClO4 was used respectively as the electrolyte, highlighting the crucial role of electrolytes in process efficiency. Both radical and non-radical mechanisms participated in the destruction of EE2, however the role of the photogenerated carriers was dominant. Ecotoxicity using Vibrio fischeri as an indicator was decreased, although not proportionately to the parent compound EE2 in the combined Solar/TiO2/WO3/PMS system.

Embryo developmental toxicity in marine medaka (Oryzias melastigma) due to parental and embryonic 17α-ethinylestradiol exposure

The synthetic estrogen 17α-ethinylestradiol (EE2) is a common component of hormone therapy and oral contraceptives and has been widely used for nearly 60 years. Numerous studies have shown that exposure to EE2 can affect embryonic development in a number of fish species. The effects of parental and embryonic EE2 exposure on embryo developmental toxicity and the underlying molecular mechanisms, however, have rarely been examined. In this study, embryos collected from parental EE2-exposed adult fish were examined to assess EE2-induecd toxicity during embryo development. The rate of embryo development including heart rate, hatching rate, and larval locomotion were measured to assess embryo developmental toxicity. The embryonic transcriptome was used to delineate the related developmental toxicity pathways. Our results suggest that parental and embryonic EE2 exposure resulted in growth retardation including a reduction in embryo heart rate, a delay in the appearance eye pigmentation, decreased hatching rate and impaired larval locomotion. In addition, gene ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Ingenuity Pathway Analysis (IPA) of transcriptome revealed that these impairments are controlled by estrogen receptor and related to eye structure, neuronal and synaptic structure, and behaviour. The key factors identified, including PRKAA2, APOB, EPHB2, OXTR, NR2E3, and POU4F2, could serve as biomarkers for assessing EE2-induced embryo developmental toxicity. For the first time, our results show that eye pigmentation is a potentially sensitive marker of EE2-induced embryo developmental toxicity.

The simpler the better: Highly sensitive 17α-ethinylestradiol sensor based on an unmodified carbon paper transducer

The remarkable features of a carbon fiber paper sensor (CP) were employed for detection of the estrogenic hormone 17α-ethinylestradiol (EE2), considered a contaminant of emerging concern due to its potential ecotoxicity and widespread in the aquatic ecosystems. In this work, an unpreceded CP pre-treatment study was conducted with the (Il)-hexacyanoferrate(III) ion pair, however a bare sensor without pre-treatment revealed higher efficiency on the oxidation of EE2 compared to a chemical and electrochemical pre-treated CP and a gold nanoparticles modified CP, being thus selected for EE2 determinations. The analytical conditions were thoroughly optimized in terms of electrolyte pH (pH 7), differential pulse voltammetry parameters (modulation time 0.003 s, amplitude 0.09 V, interval time 0.1 s and step potential 0.01 V), and analyte preconcentration potential (0.4 V) and time (180 s). The hormone can be determined by the CP in a wide linear range from 0.1 to 1000 nM, achieving a detection limit of 0.14 ± 0.005 nM and an outstanding sensitivity of 1636 ± 232 μA μM−1 cm−2 in the lowest linear zone (0.1–1 nM). The sensor was validated in river water and fish reaching good recoveries (91.2 ± 4.6 to 109.0 ± 7.1%), reproducibility and repeatability. Moreover, the sensor showed high selectivity to EE2 in the presence of several potential interfering compounds and frequently prescribed drugs, though it could not discriminate the similar hormone, 17β-estradiol, being the total concentration obtained in this case. CP-based sensors emerge as efficient electroanalytical tools, suggesting that modification of the surface may not always be beneficial in terms of sensitivity.

Synthetic estrogen bioaccumulates and changes the behavior and biochemical biomarkers in adult zebrafish

Estrogen is considered to be an endocrine disrupter and is becoming increasingly more prevalent in the daily life of humans. In some cases, estrogen is not fully metabolized by organisms and may be excreted in either its original form or in organic complex forms, into water residue systems reaching concentrations of 0.05 ng.L−1 to 75 ng.L−1. However, estrogen 17α-ethinylestradiol (EE2), which is used in oral contraceptives, is very difficult to remove from water. Here, we evaluated whether the synthetic hormone, EE2, affects the nervous system and the behavior of adult zebrafish. We established a range of concentrations (0.05, 0.5, 5, 50, and 75 ng.L−1), in addition to the control, to evaluate the effect of this compound and its bioaccumulation in zebrafish tissues. Here we show that EE2 bioaccumulates in fish and can change its behavior with an increased time in the upper zone (novel tank test) and far from the shoal segment (social preference test), demonstrating a clear anxiolytic pattern. The anxiolytic effect of EE2 can be harmful as it can affect the stress response of the species. The results presented herein reinforce the idea that the presence of EE2 in environmental water can be dangerous for non-target animals.

A sensitive, robust method for determining natural and synthetic hormones in surface and wastewaters by continuous solid-phase extraction–gas chromatography–mass spectrometry

Over recent decades, steroidal estrogens have become an emerging and very serious issue as they pose a serious threat to living organisms, soil, plants, and water resources in general. Estrogens have therefore been the subject of considerable scientific attention in order to develop new methodologies for its determination, being able of detecting them at very low concentrations. Those procedures minimize or eliminate the consumption of organic solvents and reagents that may be incompatible with the environment. In this respect, we developed a sensitive, selective method for the simultaneous determination of thirteen natural and synthetic hormones present at the nanogram-per-liter level in various types of water by using continuous solid-phase extraction in combination with gas chromatography and mass spectrometry (GC–MS). The target analytes were preferentially sorbed on an Oasis HLB sorbent column (80 mg) and eluted with acetone (600 µL) for derivatization with a mixture of 70 µL of N,O-bis(trimethylsilyl) trifluoroacetamide and trimethylchlorosilane and 35 µL of petroleum ether in a household microwave oven at 200 W for 4 min. Under optimum conditions, the ensuing method exhibited good linearity (r ≥ 0.998), good precision (RSD ≤ 7%), high recoveries (92–103%), and low detection limits (0.01–0.3 ng L−1). The method outperforms existing alternatives in robustness, sensitivity, throughput, flexibility—it allows both estrogens, progestogens, and androgens to be determined simultaneously—and compliance with the principles of Green Chemistry. It was successfully used to analyze various types of water samples (mineral, tap, well, pond, swimming pool, river, and waste) that were found to contain four estrogens (estrone, 17β-estradiol, 17α-ethinylestradiol, and hexestrol), two progestogens (testosterone, dihydrotestosterone), and one progestogen (progesterone) at concentrations ranging from 3.0 to 110 ng L−1.

Metabolic and oxidative status alterations induced in Ruditapes philippinarum exposed chronically to estrogen 17α-ethinylestradiol under a warming scenario.

The presence of pharmaceuticals in the aquatic environment is an ongoing concern. However, the information regarding their effects under different climate change scenarios is still scarce. 17α-ethinylestradiol (EE2) is widely present in different aquatic systems showing negative impacts on aquatic organisms even when present at trace concentrations (≈1ng/L). Nevertheless, its impact on bivalves is poorly understood, especially considering the influence of climate change factors. This study aimed to assess the toxicological impacts of EE2 under current and predicted warming scenarios, in the edible clam Ruditapes philippinarum. For this, clams were exposed for 28 days to different EE2 concentrations (5, 25, 125, 625ng/L), under two temperatures (17 °C (control) and 21 °C). Drug concentrations, bioconcentration factors and biochemical parameters, related to oxidative stress and energy metabolism, were evaluated. Results showed that under actual and predicted temperature scenarios EE2 concentrations led to a disturbance in redox homeostasis of the clams, characterized by an increase in oxidized glutathione in contaminated organisms compared to control ones. Nevertheless, clams were capable to cope with the stressful conditions, activating their defence mechanisms (especially at the highest exposure concentration and in particular at increased temperature), and no oxidative damage occured. Although limited effects were observed, the present findings indicate that under both temperatures contaminated clams altered their biochemical performance, which can impair their sensitivity and protection capacity to respond to other environmental changes and/or affect their capacity to grow and reproduce. The results presented here highlight the need for further research on this thematic, considering that climate change is an ongoing problem, and the levels of some pharmaceutical drugs will continue to increase in marine/estuarine environments.

Estrogens as immunotoxicants: 17α-ethinylestradiol exposure retards thymus development in zebrafish (Danio rerio).

Estrogenic endocrine disrupting compounds (EEDCs) can cause alterations in sexual development and reproductive function of fish. Growing evidence suggests that EEDCs can also interfere with development and function of innate immunity of fish. The present study examined a potential disruptive effect of EEDCs at field-relevant concentrations on the development of adaptive immunity, more specifically the thymus. Zebrafish (Danio rerio) were exposed from fertilization until 64 days post-fertilization (dpf) to environmentally relevant (3 and 10ng/L) concentrations of the synthetic estrogen 17α-ethinylestradiol (EE2). The exposure duration covered the period of initial thymus differentiation to maximum growth. Thymus development was assessed by histological and morphometric (thymus area) analysis, thymocyte number, and transcript levels of thymocyte marker genes. Additionally, transcript levels of the estrogen receptors (esr1 and esr2a) were determined. The EE2 exposure altered sexual development (gonad differentiation, transcript levels of hepatic vitellogenin and estrogen receptors) of zebrafish, as expected. At the same time, the EE2 treatment reduced the thymus growth (thymus area, thymocyte number) and transcript levels of thymus marker genes. The expression of the thymic estrogen receptors responded to the EE2 exposure but in a different pattern than the hepatic estrogen receptors. After the 64-day-exposure period, the juvenile fish were transferred into clean water for another 95 days to assess the reversibility of EE2-induced effects. The thymic alterations were found to be reversible in female zebrafish but persisted in males. The present study provides the first evidence that the development of the fish adaptive immune system is sensitive to EEDCs, and that this takes place at concentrations similar to those that disrupt sexual development.

Drug Interactions Involving 17α-Ethinylestradiol: Considerations Beyond Cytochrome P450 3A Induction and Inhibition.

It is widely acknowledged that drug-drug interactions (DDIs) involving estrogen (17α-ethinylestradiol (EE))-containing oral contraceptives (OCs) are important. Consequently, sponsors of new molecular entities (NMEs) often conduct clinical studies with priority given to OCs as victims of cytochrome P450 (CYP) 3A (CYP3A) induction and inhibition. Such scenarios are reflected in the US Food and Drug Administration-issued guidance documentation related to OC DDI studies. Although CYP3A is important, OCs such as EE are metabolized by sulfotransferase 1E1 and UDP-glucuronosyltransferase (UGT) 1A1, expressed in the gut and liver, and so both can also serve as loci of victim OC DDI. Therefore, for any NME, one should carefully consider its induction and inhibition profile involving CYP3A4/5, UGT1A1, and SULT1E1. As DDI perpetrators, available clinical DDI data indicate that EE-containing OCs can induce (e.g., UGT1A4 and CYP2A6) and inhibit (CYP1A2 ≥ CYP2C19 > CYP3A4/5 > CYP2C8, CYP2B6, CYP2D6, and CYP2C9) various CYP forms. Although available in vitro CYP inhibition data do not explain such a graded inhibitory effect in vivo, it is hypothesized that EE differentially modulates CYP expression via potent agonism of the estrogen receptor expressed in the gut and liver. From the standpoint of the NME as potential OC DDI victim, therefore, it is important to assess its projected (pre-phase I) or known therapeutic index and pharmacokinetic profile (fraction absorbed, absolute oral bioavailability, clearance/extraction class, fraction metabolized by CYP1A2, CYP2C19, CYP2A6, and UGT1A4). Such information can enable the prioritization, design, and interpretation of NME-OC DDI studies.

Behavioral and reproductive effects in Poecilia vivipara males from a tropical estuary affected by estrogenic contaminants

Contamination of aquatic habitats by endocrine disruptor chemicals is a major concern globally. This study evaluated histochemical, behavioral, and reproductive effects on adult male Poecilia vivipara sampled from Capibaribe River Estuarine System (CRES), compared to laboratory control males after breeding with virgin control females. CRES is contaminated by a mixture of estrogenic contaminants estrone, 17β-estradiol, estriol, 17α-Ethinylestradiol, bisphenol A and caffeine in concentrations averaging 13.9; 4.2; 19.5; 8.6; 27 and 23.2 ng L−1, respectively. Estrogenic risk in 17β-estradiol-equivalent-concentrations is above probable no effect concentrations. Males sampled from CRES indicated liver phosphoprotein induction, decreased number of contacts and copulation attempts when paired with control females, slower swimming speed and lower female impregnation success rates, compared to control males. A reduction of 62% in fecundity was observed in control females paired with field sampled males compared with control males. Our results highlight hazards posed to fish reproduction by estrogenic micropollutants.

Gonadal sex steroid hormone secretion after exposure of male rats to estrogenic chemicals and their combinations

Environmental chemicals can interfere with the endocrine axis hence they are classified as endocrine disrupting chemicals (EDCs). Bisphenol S (BPS) is used in the manufacture of consumer products because of its superior thermal stability and is thought to be a safe replacement chemical for its analog bisphenol A (BPA). However, the safety profile of these compounds alone or in the presence of other EDCs is yet to be fully investigated. Also, the estrogenic chemical 17α-ethinyl estradiol (EE2) and a constituent of female oral contraceptives for women, is present in water supplies. To simulate concurrent exposure of the population to chemical mixtures, we investigated the effects of BPA, BPS, EE2, and their combinations on sex steroid secretion in the growing male rat gonad. Prepubertal and pubertal male rats at 21 and 35 days of age were provided test chemicals in drinking water (parts per billion) for 14 days. At termination of exposure, some individual chemical effects were modified by exposure to chemical combinations. Single chemical exposures markedly decreased androgen secretion but their combination (e.g., BPA + BPS + EE2) caused the opposite effect, i.e., increased Leydig cell T secretion. Also, the test chemicals acting alone or in combination increased testicular and Leydig cell 17β-estradiol (E2) secretion. Chemical-induced changes in T and E2 secretion were associated with altered testicular expression of the cholesterol side-chain cleavage (Cyp11a1) and 17β-hydroxysteroid dehydrogenase (Hsd17β) enzyme protein. Additional studies are warranted to understand the mechanisms by which single and chemical combinations impact function of testicular cells and disrupt their paracrine regulation.

Health Effects and Life Stage Sensitivities in Zebrafish Exposed to an Estrogenic Wastewater Treatment Works Effluent.

A wide range of health effects in fish have been reported for exposure to wastewater treatment work (WwTW) effluents including feminized responses in males. Most of these exposure studies, however, have assessed acute health effects and chronic exposure effects are less well established. Using an Estrogen Responsive Element-Green Fluorescent Protein (ERE-GFP)-Casper transgenic zebrafish, we investigated chronic health effects and life stage sensitivities for exposure to an estrogenic WwTW effluent and the synthetic estrogen 17α-ethinylestradiol (EE2). Exposure to the WwTW effluent (at full strength;100%) and to 10 ng/L (nominal) EE2 delayed testis maturation in male fish but accelerated ovary development in females. Exposure to 50% and 100% effluent, and to 10 ng/L EE2, also resulted in skewed sex ratios in favor of females. Differing patterns of green fluorescent protein (GFP) expression, in terms of target tissues and developmental life stages occurred in the ERE-GFP- zebrafish chronically exposed to 100% effluent and reflected the estrogenic content of the effluent. gfp and vitellogenin (vtg) mRNA induction were positively correlated with measured levels of steroidal estrogens in the effluent throughout the study. Our findings illustrate the importance of a fish’s developmental stage for estrogen exposure effects and demonstrate the utility of the ERE-GFP zebrafish for integrative health analysis for exposure to estrogenic chemical mixtures.

Efficacy and reusability of mixed-phase TiO2–ZnO nanocomposites for the removal of estrogenic effects of 17β-Estradiol and 17α-Ethinylestradiol from water

Photocatalytic removal of estrogenic compounds (ECs), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2) were assessed using a TiO2–ZnO nanocomposite (NC) over a range of initial EC concentration (Co; 10 mg/L – 0.05 mg/L). Photocatalytic removal was evaluated under UV and visible irradiation using 10 mg/L NC over 240 min duration. After 240 min, analysis using GCxGC TOF MS revealed 100% transformation at Co ≤ 1 mg/L and ≥25% transformation at Co ≤ 10 mg/L under visible irradiation. Degradation was accompanied by breakdown of the fused ring structure of E2, generating smaller molecular weight by-products which were subsequently mineralized as revealed through TOC removal. With UV photocatalysis, ~30% and ~20% mineralization was attained for E2 and EE2, respectively, for Co of 10 mg/L. Under visible irradiation, ~25% and ~10% mineralization was achieved for E2 and EE2, respectively. Estrogenicity variation was estimated using the E-screen assay conducted with estrogen receptor-positive MCF-7 breast cancer cells. Complete removal of estrogenicity of ECs was confirmed after 240 min of photocatalysis under UV and visible irradiation. FTIR spectroscopy-based analysis of the NC after E2 photocatalysis revealed the presence of sorbed organics. Desorption, followed by GC × GC TOF-MS analysis revealed these organics as by-products of photocatalysis. Desorption of sorbed organics followed by recalcination at 600 °C for 1 h regenerated the active sites on the NC, enabling its efficient reuse for 3 cycles under visible irradiation without loss in activity.

Vitellogenin gene expression in marine mussels exposed to ethinylestradiol: No induction at the transcriptional level

Vitellogenin (Vtg), a large multidomain protein precursor of egg-yolk proteins, is used as an endocrine disruption biomarker in fish, and in the last decades, its use has been extended to invertebrates like mollusks. However, it remains unclear whether invertebrate endocrine system produces Vtg in response to estrogens, like it occurs in oviparous vertebrates. In a previous study, no evidence of induction of Vtg expression at protein level was found in gonads of the marine mussel Mytilus galloprovincialis after exposure to the estrogenic chemical 17α-ethinylestradiol (EE2). In the present follow-up study, it was investigated whether there is any effect of EE2 on Vtg abundance at transcriptional level in M. galloprovincialis gonads. To this aim, RT-qPCR analysis targeting three different domains of Vtg transcript was performed on gonads of mussels that were exposed either 4 or 24 days to 100 ng/L EE2. In addition, several reference genes were analysed and a selection of these for potential use in further RT-qPCR analyses on mussel male and female gonads is provided. Results showed higher expression in females than in males for the three analysed Vtg domains, and no evidence of Vtg mRNA induction due to EE2 either in females or males. The present results, together with those obtained from previous analysis at protein level, support that Vtg is not an adequate biomarker for xenoestrogenicity in marine mussels. Additionally, nucleotide sequences of Vtg transcripts of three closely-related species from Mytilus edulis complex (M. galloprovincialis, M. edulis and M. trossulus) are provided and compared with Vtg sequences from other mollusk species to assess the level of conservation and evolutionary relationships among species.

Biodegradation of natural and synthetic endocrine-disrupting chemicals by aerobic granular sludge reactor: Evaluating estrogenic activity and estrogens fate.

In this study, the biodegradation of endocrine-disrupting chemicals (EDCs) (namely the natural and synthetic estrogens 17β-estradiol (E2) and 17α-ethinylestradiol (EE2), respectively) was assessed in an aerobic granular sludge (AGS) sequencing batch reactor (SBR) treating simulated domestic sewage. To better understand the fate of these compounds, their concentrations were determined in both liquid and solid (biomass) samples. Throughout the operation of the reactor, subjected to alternating anaerobic and aerated conditions, the removal of the hormones, both present in the influent at a concentration of 20μgL-1, amounted to 99% (for E2) and 93% (for EE2), with the latter showing higher resistance to biodegradation. Through yeast estrogen screen assays, an average moderate residual estrogenic activity (0.09μgL-1 EQ-E2) was found in the samples analysed. E2 and EE2 profiles over the SBR cycle suggest a rapid initial adsorption of these compounds on the granular biomass occurring anaerobically, followed by biodegradation under aeration. A possible sequence of steps for the removal of the micropollutants, including the key microbial players, was proposed. Besides the good capability of the AGS on EDCs removal, the results revealed high removal efficiencies (>90%) of COD, ammonium and phosphate. Most of the incoming organics (>80%) were consumed under anaerobic conditions, when phosphate was released (75.2 mgP L-1). Nitrification and phosphate uptake took place along the aeration phase, with effluent ammonium and phosphate levels around 2mgL-1. Although nitrite accumulation took place over the cycle, nitrate consisted of the main oxidized nitrogen form in the effluent. The specific ammonium and phosphate uptake rates attained in the SBR were found to be 3.3 mgNH4+-N gVSS-1.h-1 and 6.7 mgPO43--P gVSS-1 h-1, respectively, while the specific denitrification rate corresponded to 1.0 mgNOx--N gVSS-1 h-1.

Occurrence and distribution of estrogenic substances in the northern South China Sea

Estrogenic substances are today among the contaminants of emerging concern. Besides naturally occurring estrogens, other natural and synthetic substances can mimic a hormonal action due to their structural resemblance to hormones, possibly affecting the endocrine system of living organisms. Estrogens have been detected in inland water bodies such as influents and effluents of waste water treatment plants as well as in rivers, but data on their distribution and variability in the marine ecosystem are still limited. Surface water samples obtained during two research cruises on the northern shelf of the South China Sea (SCS) near the Pearl River Estuary, in September 2018 and in August 2019, were investigated for estrogenic substances, namely estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), genistein (GEN), daidzein (DAI) and zearalenone (ZEN). Among the target analytes, the natural hormones E1 and E2, as well as the synthetic EE2, were the most abundant with maximum concentrations of 1.1 ng L−1, 0.7 ng L−1 and 0.6 ng L−1, respectively. Of substances produced by plants and fungi, GEN, DAI and ZEN, only GEN was detected (1.2 ng L−1). High concentrations occurred predominantly close to the coast, which was also reflected in the calculated estradiol equivalent quotients (up to 1.4 ng L−1). In general, the distribution of estrogenic substances observed in both years shows a regional and inter-annual variability consistent with the modeled surface current data for the SCS. Regarding single estrogenic compounds and estradiol equivalents, marine organisms in the northern SCS might be exposed to high potential risk.