Assessing estrogenic activity in surface water and sediment of the Liao River system in northeast China using combined chemical and biological tools

This paper investigated some selected estrogenic compounds (4-t-octylphenol: 4-t-OP; 4-nonylphenols: 4-NP; bisphenol-A: BPA; diethylstilbestrol: DES; estrone: E1; 17β-estradiol: E2; 17α-Ethinylestradiol: EE2; triclosan: TCS) and estrogenicity in the Liao River system using the combined chemical and in vitro yeast screen bioassay and assessed their ecological risks to aquatic organisms. The estrogenic compounds 4-t-OP, 4-NP, BPA, E1, E2 and TCS were detected in most of the samples, with their concentrations up to 52.1 2065.7, 755.6, 55.8, 7.4 and 81.3 ng/L in water, and up to 8.6, 558.4, 33.8, 7.9, <LOQ and 33.9 ng/g in sediment, respectively. However, DES and EE2 were not detected in the Liao River. The estrogen equivalents (EEQ) of the water and sediment samples were also measured by the bioassay. High estrogenic risks to aquatic organisms were found in the river sections of metropolitan areas and the lower reach of the river system.     

Suppression of estrogenic activity of 17-beta-estradiol by beta-cyclodextrin

The suppressive effects of cyclodextrins (CDs) on the strong estrogenic activity of 17β-estradiol (E2) in water environments were investigated in this study. Cyclodextrins are doughnut-shaped molecules that possess a hydrophobic cavity and a hydrophilic exterior. The cavity can incorporate nonpolar molecules as guests to form inclusion complexes. β-CD and 2-hydroxypropyl-β-CD (HP-β-CD) were the most successful in forming a complex with E2 and improving its low aqueous solubility. The E2/CDs complexes bound to the estrogen receptor in a cell-free system as determined by ELISA and suppressed the hormone activities as measured by a yeast two-hybrid assay. These results indicate that hydrophobic E2 is easily transported through the lipid zone of the plasma membrane into the target cell and can bind to the nuclear receptor. However, the hydrophilic E2/β-CD and E2/HP-β-CD complexes do not penetrate the membrane. Therefore, these CDs are able to suppress the hormone activity of E2 through complex formation.     

Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.     

Occurrence and photochemical degradation of 17alpha-ethinylestradiol in Acushnet River Estuary

17alpha-Ethinylestradiol (EE2), a major constituent of common contraceptive pills, and three other estrogenic hormones, estrone (E1), 17beta-estradiol (E2) and mestranol (MeEE2) have been determined in Acushnet River Estuary seawater using a GC-MS technique. Among three estrogenic compounds detected, EE2 has the highest concentration, up to 4.7 ng/l, at which EE2 may affect lobster and other fish abundance in the coastal seawater due to its high biological activity on fish feminization. Two natural estrogenic hormones, E1 and E2 have also been found in the estuary at concentrations up to 1.2 ng/l and 0.83 ng/l, respectively. Although EE2 is persistent to microbial degradation, it can undergo a rapid photodegradation in estuarine seawater under natural sunlight irradiation, with a half-life of less than 1.5 days in spring sunny days.     

Effect of certain chemicals on the reproduction of medaka (Oryzias latipes)

In order to understand the effects of estrogenic chemicals on fish reproduction, we exposed male medaka (Oryzias latipes) to a natural estrogen [17 beta-estradiol (17 beta-E2)] and three estrogenic chemicals [bisphenol-A, nonylphenol (NP) and di(2-ethylhexyl)phthalate (DEHP)]. After two weeks' exposure, one male medaka was kept together with two female medaka for spawning, and the number of eggs and hatchings were compared to those of a negative control group. The results indicated that exposure to 17 beta-E2 caused a significant decrease in the number of eggs and hatchings as compared to the negative control group at and above 3 nmol/l. Also, the highest concentrations of bisphenol-A and NP caused a decrease in the number of hatchings, but no decrease in hatchings was observed in DEHP treatments. In the treatment using these chemicals the decrease in egg numbers was not so much as in hatching numbers. When compared to other in vitro studies, concentrations observed to have adverse effects on reproduction in this study are generally lower. In addition, it was suggested that physical alterations, such as an induction of plasma vitellogenin, were caused at much lower concentrations than those at which a decline in reproductivity was actually induced.     

4-Alkylphenols and related chemicals show similar effect on the function of human and rat estrogen receptor alpha in reporter gene assay

Alkylphenols (APs) are widely used as important industrial materials and have attracted lots of attention because of their potential estrogenic activities. In this study, we developed human estrogen receptor alpha (hERalpha) and rat estrogen receptor alpha (rERalpha) mediated reporter gene assays and compared the estrogenic activity of APs and related chemicals based on the two ERalpha. Human breast cancer cell line MCF-7 was co-transfected with Gal4-fused hERalpha and corresponding reporter plasmid; African green monkey kidney cell line CV-1 was co-transfected with rERalpha and reporter gene. Both assays showed acceptable response to natural estrogen 17beta-estradiol (E2) with EC50 of 0.16 nM and 4.7 nM. Then the estrogenic activity of 4-APs, 4-phenylphenol and bisphenol-A were evaluated and compared with the effects of E2. The data suggested that test APs and related chemicals possessed weakly estrogenic activity and the activity of test APs increased with the increase of substituent size. This structure-activity relationship helped to infer the activity of chemicals with similar feature. Furthermore, test APs showed similar effect on the function of hERalpha and rERalpha. This consistency helped to extrapolate in vivo rodent data to human being when performing risk assessment of endocrine disruptors.     

How seasonality affects the flow of estrogens and their conjugates in one of Japan's most populous catchments

A detailed study of the free and conjugated estrogen load discharged by the eight major sewage treatment plants into the Yodo River basin, Japan was carried out. Sampling campaigns were focused on the winter and autumn seasons from 2005 to 2008 and the free estrogens estrone(E1), 17β-estradiol(E2), estriol(E3), 17α-ethynylestradiol(EE2) as well as their conjugated (sulfate and glucuronide) forms. For both sewage effluent and river water E2 and E1 concentrations were greatest during the winter period (December-March). This coincides with the period of lowest rainfall and lowest temperatures in Japan. E1 was the dominant estrogenic component in effluent (means of 10-50 ng/L) followed by E2 (means of 0.5-3 ng/L). The estrogen sulfate conjugates were found intermittently in the 0.5-1.7 ng/L concentration range in the sewage effluents. The greatest estrogen exposure was found to be in the Katsura River tributary which exceeded 1 ng/L E2-equivalents during the winter period.     

Estrogenic activity removal of 17beta-estradiol by ozonation and identification of by-products

This work investigated the degradation of a natural estrogen (17beta-estradiol) and the removal of estrogenic activity by the ozonation process in three different pHs (3, 7 and 11). A recombinant yeast assay (YES assay) was employed to determine estrogenic activity of the ozonized samples and of the by-products formed during the ozonation. Ozonation was very efficient for the removal of 17beta-estradiol in aqueous solutions. High removals (>99%) were achieved with low ozone dosages in the three different pHs. Several by-products were formed during the ozonation of 17beta-estradiol. However, only a few compounds could be identified and confirmed. Different by-products are formed at different pHs, which is probably due to different chemical pathways and different oxidants (O(3) and OH radical). The by-products formed at pH 11 were 10epsilon-17beta-dihydroxy-1, 4-estradieno-3-one (DEO) and 2-hydroxyestradiol, which were not formed in pH 3. Only testosterone could be observed in pH 3, whereas at pH 7 all three by-products were found. At pH 7 and 11 the applied ozone dosages were not enough to remove all the estrogenicity from samples, even though the 17beta-estradiol residual concentration for these two pHs was lower than at pH 3. Higher estrogenicity was detected at pH 11. An explanation to this fact may be that oxidation via OH radical forms more by-products with estrogenic activity. Probably, the formation of 2-hydroxyestradiol at pHs 7 and 11 is contributing to the residual estrogenicity of samples ozonized at these pHs. In this work, complete removal of estrogenic activity was only obtained at pH 3.     

Biochemical and biological assays of endocrine disrupting compounds in various manure matrices

Wastes generated by animal agriculture have complex matrices and present a significant challenge for achieving accurate measurements of estrogens. The objective of this study was to compare two different extraction methods and two different biochemical and biological estrogen assays on two dairy manure matrices. The two extraction methods studied in this research were a two-step base-toluene extraction and a one-step cyclohexane extraction. The two assays assessed were an enzyme-linked immunosorbent assay (ELISA) and a yeast estrogen screen (YES) bioassay. Estrogenic activity was measured directly as 17beta-estradiol (E2) through the ELISA method or as E2 equivalents (E2-eq) through the YES bioassay. Both extraction methods yielded reasonable recoveries from distilled water in the absence of matrix interferences. In manure samples, ELISA resulted in recoveries slightly higher than 100% in two types of dairy manures, but YES recoveries varied. The YES bioassay detected much higher estrogenic activities in dairy manure compared to the E2 concentrations measured by ELISA. The base-solvent extraction yielded higher E2 concentrations in dairy manure compared to the one-step cyclohexane extraction. These results suggest that manure matrices vary sufficiently that extraction methods must be optimized for specific assays utilized to quantify estrogens in manures.     

Estrogenic effects of dissolved organic matter and its impact on the activity of 17β-estradiol

Background and purpose  

The ubiquitous dissolved organic matter (DOM) is actually not inert as we always think, and the hormone-like effects of DOM have been reported. The objective of this study was to investigate the estrogenic effects of DOM and its impact on the activity of the natural estrogen 17β-estradiol (E2).     

Occurrence and change of estrogenic activity in the process of drinking water treatment and distribution

From 2010 to 2012, the Yangtze River and Hanjiang River (Wuhan section) were monitored for estrogenic activities during various water level periods. Using a recombinant yeast estrogen screen (YES) assay, 54 water samples were evaluated over the course of nine sampling campaigns. The mean 17β-estradiol equivalent (EEQ) value of raw water from the Yangtze River was 0–5.20 ng/L; and the EEQ level from the Hanjiang River was 0–3.22 ng/L. In Wuhan, drinking water treatment plants (DWTPs) using conventional treatments reduced estrogenic activities by more than 89 %. In general, water samples collected during the level period showed weaker estrogenic activities compared to those collected during the dry period. The samples collected in 2010 showed the strongest estrogenic activities of the 3-year period. The lack of correlations between estrogenic activities and selected common water quality parameters showed that estrogenic activity cannot be tied to common water quality parameters.     

Estrogenic and dioxin-like compounds in sediment from Zierikzee harbour identified with CALUX assay-directed fractionation combined with one and two dimensional gas chromatography analyses

The identity of compounds responsible for estrogenic and dioxin-like activities in sediment from the harbour of the small town Zierikzee in Zeeland, The Netherlands, was investigated using a bioassay directed fractionation approach with the in vitro estrogen and dioxin responsive reporter gene assays ER- and DR-CALUX. For identification of compounds exhibiting activity in the bioassays, either one or two-dimensional GC in combination with quadrupole (MSD), ion trap (ITD) or time-of-flight mass spectrometric detection (ToF-MS) was used, depending on the biological and chemical characteristics and the complexity of the fractions. The natural estrogenic hormone 17-β-estradiol and its metabolite estrone were identified with GC–ITD as the main contributors to the estrogenic activity. After successive rounds of fractionation, the dioxin-like activity could be explained by the presence of various polycyclic aromatic hydrocarbons identified with GC–MSD and two-dimensional comprehensive GC × GC–ToF-MS. Some estrogenic activity of a relatively non-polar nature remained unidentified.     

Endocrine disrupting activities in sewage effluent and river water determined by chemical analysis and in vitro assay in the context of granular activated carbon upgrade

As part of endocrine disruption in catchments (EDCAT) programme, this work aims to assess the temporal and spatial variations of endocrine disrupting chemicals (EDCs) in River Ray, before and after the commissioning of a full-scale granular activated carbon (GAC) plant at a sewage treatment works (STW). Through spot and passive sampling from effluent and river sites, estrogenic and anti-androgenic activities were determined by chemical analysis and in vitro bio-assay. A correlation was found between chemical analyses of the most potent estrogens (estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2)) and yeast estrogen screen (YES) measurement, both showing clearly a reduction in estrogenic activity after the commissioning of the GAC plant at the STW. During the study period, the annual average concentrations of E1, E2 and EE2 had decreased from 3.5 ng L⁻¹, 3.1 ng L⁻¹ and 0.5 ng L⁻¹ to below their limit of detection (LOD), respectively, with a concentration reduction of at least 91%, 81% and 60%. Annual mean estrogenic activity measured by YES of spot samples varied from 1.9 ng L⁻¹ to 0.4 ng L⁻¹ E2 equivalent between 2006 and 2008 representing a 79% reduction. Similarly, anti-androgenic activity measured by yeast anti-androgen screen (anti-YAS) of spot samples was reduced from 148.8 to 22.4 μg flutamide L⁻¹, or by 85%. YES and anti-YAS values were related to each other, suggesting co-existence of both types of activities from chemical mixtures in environmental samples. The findings confirm the effectiveness of a full-scale GAC in removing both estrogenic and anti-androgenic activities from sewage effluent.     

Fate estimation of estrogenic substances in an urban river by flux calculation.

This study investigated the fate of estrogenic substances in an urban river receiving discharge from wastewater treatment plants (WTPs) by flux calculation, focusing on the middle reaches of the Tama River in Tokyo, which is one of the most urbanized rivers in Japan. The level of estrogenic activity flux was almost negligibly small at the upstream station. The level was considerably raised after inflows from the WTPs and then the level declined in the lower reaches of the river. When contributions of estrogenic substances to estrogenic activity were estimated, estrone (E1) was the primary contributor to the total estrogenic activity in all the sampling stations, followed by estradiol (E2). The contribution of nonylphenol to estrogenic activity was small. The E1 and E2 accounted for approximately 90% or more of estrogenic activity in the Tama River. As for the total fluxes of the estrogenic substances in the study area in the Tama River, the proportion of flux associated with WTP discharge was approximately 100% of the total fluxes, and the effects of the tributaries flowing into the river were almost negligible. When the reduction ratios of estrogenic activity were calculated by the flux, the ratios were found to increase toward the lower reaches of the river. Similar changes were observed for E1. Meanwhile, the change of reduction ratios for E2 was different from that observed for estrogenic activity.     

Estrogen equivalent concentration of 13 branched para-nonylphenols in three technical mixtures by isomer-specific determination using their synthetic standards in SIM mode with GC-MS and two new diasteromeric isomers

Thirteen isomers of branched para-nonylphenols (para-NP) in three technical mixtures were isomer-specifically determined using their synthesized standards by SIM of structurally specific ions, m/z 135, 149 or 163 with GC–MS. Of the 13 isomers, four isomers, 4-(2,4-dimethylheptan-4-yl)phenol, 4-(4-methyloctan-4-yl)phenol, 4-(3-ethyl-2-methylhexan-2-yl)phenol (3E22NP) and 4-(2,3-dimethylheptan-2-yl)phenol synthesized for their determinations were first used as standard substances. The 13 isomers in the technical mixtures individually occurred at mass percent portion of more than 2%. The total mass percent portions in the mixtures from Tokyo Chemical Industry (TCI), Aldrich, and Fluka covered with 89 ± 2%, 75 ± 4% and 77 ± 2%, respectively. The abundance of 4-(3,6-dimethylheptan-3-yl)phenol in the three mixtures was the largest with 11.1 ± 2% to 9.9 ± 0.3%, while that of 4-(2-methyloctan-2-yl)phenol was the smallest with 2.9 ± 0.3% to 3.0 ± 0.2%. Additionally, structures of four new isomers of more than 1% portion present in a technical mixture were elucidated as two pairs of diastereomeric isomers: two types of 4-(3,4-dimethylheptan-4-yl)phenol (344NP) and those of 4-(3,4-dimethylheptan-3-yl)phenol (343NP). By estrogenic assay of 13 isomers with yeast estrogen screen system, the activity of 3E22NP was the highest, while that of 4-(3-methyloctan-3-yl)phenol was the least. Their relative activities to that of 3E22NP were individually calculated. Estrogenic equivalent concentrations of the three technical mixtures were predictively evaluated. The ratio of the EEC to the conventional concentration, total mass percent portions of the 13 isomers in technical mixtures were 0.208 for TCI, 0.206 for Aldrich and 0.205 for Fluka. The predicted estrogenic activity of measured concentration of para-NP in technical mixtures was approximately 5-fold greater than the measured estrogen agonist activity.     

Bioanalytical and instrumental analysis of estrogenic activities in drinking water sources from Yangtze River Delta

The estrogenic activities of source water from Yangtze River, Huaihe River, Taihu Lake and groundwater in Yangtze River Delta in the dry and wet season were determined by use of reporter gene assays based on African green monkey kidney (CV-1) cell lines. Higher estrogenic activities were observed in the dry season, and the estrogenic potentials in water samples from Taihu Lake were greater than other river basins. None of the samples from groundwater showed estrogen receptor (ER) agonist activity. The 17β-Estradiol (E2) equivalents (EEQs) of water samples in the dry season ranged from 9.41 × 10^?1 to 1.20 × 10¹ ng E2 L^?1. In the wet season, EEQs of all the water samples were below the detection limit as 9.00 × 10^?1 ng E2 L^?1 except for one sample from Huaihe River. The highest contribution of E2 was detected in Yangtze River as 99% of estrogenic activity. Nonylphenol (NP, 100% detection rate) and octylphenol (OP, 100% detection rate) might also be responsible for the estrogenic activities in water sources. Potential health risk induced by the estrogenic chemicals in source water may be posed to the residents through water drinking.     

Occurrence of free estrogens,conjugated estrogens,and bisphenol A in fresh livestock excreta and their removal by composting in North China

An efficient pretreatment and analytical method was developed to investigate the occurrence and fate of four free estrogens (estrone (E1), 17β-estradiol (17β-E2), estriol (E3), and 17α-ethinylestradiol (EE2)), four conjugated estrogens (estrone-3-sulfate sodium salt (E1-3S), 17β-estradiol-3-sulfate sodium salt (E2-3S), estrone-3-glucuronide sodium salt (E1-3G), and 17β-estradiol-3-glucuronide sodium salt (E2-3G)), and bisphenol A (BPA) in three livestock farms raising beef cattle, cows, sheep, swine, and chickens in Qi County, which is located in North China. The results demonstrated that one cow and one beef cattle excreted 956.25–1,270.41 and 244.38–319.99 μg/day of total (free and conjugated) estrogen, respectively, primarily through feces (greater than 91 %), while swine excreted 260.09–289.99 μg/day of estrogens, primarily through urine (98–99 %). The total estrogen excreted in sheep and broiler chicken feces was calculated to be 21.64–28.67 and 4.62–5.40 μg/day, respectively. It was determined that conjugated estrogens contributed to 21.1–21.9 % of the total estrogen excreted in cow feces and more than 98 % of the total estrogen excreted in swine urine. After composting, the concentration of total estrogen decreased by 18.7–59.6 %; however, increased levels of BPA were measured. In treated compost samples, estrogens were detected at concentrations up to 74.0 ng/g, which indicates a potential risk of estrogens entering the surrounding environment.     

AhR-mediated and antiestrogenic activity of humic substances

Humic substances (HS) were for decades regarded as inert in the ecosystems with respect to their possible toxicity. However, HS have been recently shown to elicit various adverse effects generally attributed to xenobiotics. In our study, we used MVLN and H4IIE-luc cell lines stably transfected with luciferase gene under control of estrogen receptor (ER) and Ah receptor (AhR; receptor connected with so-called dioxin-like toxicity) for assessment of anti/estrogenic and AhR-mediated effects of 12 commercially available humic substances. Out of those, five humic acids were shown to induce AhR-mediated activity with relative potencies related to TCDD 2.6 x 10(-8)-7.4 x 10(-8). Organic extracts of HS solutions also elicited high activities what means that lipophilic molecules are responsible for a great part of effect. However, relatively high activity remaining in extracted solution suggests also presence of polar AhR-agonists. Contribution of persistent organic compounds to the observed effects was ruled out by H(2)SO(4) treatment. Eight out of twelve HS elicited significant antiestrogenic effects with IC(50) ranging from 40 to 164 mg l(-1). The possible explanations of the antiestrogenic effect include sorption of 17-beta-estradiol (E2) on HS, changes in membrane permeability for E2 or another specific mechanism.