Steroid estrogens, conjugated estrogens and estrogenic activity in farm dairy shed effluents

Agricultural wastes are a source of steroid estrogens and, if present, conjugated estrogens may add to the estrogen load released to soil and aquatic environments. Dairy shed effluent samples were collected from 18 farms for analysis of steroid estrogens by GC-MS, conjugated estrogens by LC-MS-MS, and estrogenic activity by E-screen in vitro bioassay. 17α-estradiol was found at highest concentrations (median 730 ng l⁻¹), followed by estrone (100 ng l⁻¹) and 17β-estradiol (24 ng l⁻¹). Conjugated estrogens (estrone-3-sulfate, 17α-estradiol-3-sulfate and 17β-estradiol-3,17-disulfate) were measured in most samples (12-320 ng l⁻¹). Median estrogenic activity was 46 ng l⁻¹ 17β-estradiol equivalents. Conjugated estrogens contributed up to 22% of the total estrogen load from dairy farming, demonstrating their significance. Steroid estrogens dominated overall estrogenic activity measured in the samples. Significantly, 17α-estradiol contributed 25% of overall activity, despite potency 2% that of 17β-estradiol, highlighting the importance in environmental risk assessments of this previously neglected compound.     

Horizontal distribution of steroid estrogens in surface sediments in Tokyo Bay

A monitoring survey was conducted to investigate the distribution of steroid estrogens and their conjugates in surface sediments of Tokyo Bay for the first time, which is known as one of the most heavily polluted marine embayments in the world. The surface sediment samples were collected at 20 locations covering the whole area of Tokyo Bay and analyzed for steroid estrogens and their conjugates using liquid chromatography-tandem mass spectrometry. The concentrations of 17beta-estradiol (beta-E2) and estrone (E1) ranged from ND (below the detection limit; <0.07) to 0.59 and from 0.05 to 3.60 ng g-1 dry, respectively. Those concentrations were higher in the northern part of the bay, which is directly receiving huge quantities of pollutants from adjacent rivers containing a large amount of municipal and industrial wastewater. 17alpha-Estradiol (alpha-E2) and estrone-3-sulfate (E1-3S) were detected in some of the samples, whereas neither of the other conjugates, estriol (E3) or 17alpha-ethynylestradiol (EE2), was found.     

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.     

The arrival and discharge of conjugated estrogens from a range of different sewage treatment plants in the UK

The occurrence of free and conjugated estrogens was examined in a survey of eleven sewage treatment plants (STPs) and their discharge water in the United Kingdom using grab sampling. The STPs included trickling filter with and without tertiary treatment, and activated sludge with tertiary treatment. For three activated sludge plants both influent and effluent samples were compared. For a further 8 STPs only the effluent was examined. The estrone-3-sulphate, estradiol-3-sulphate and estriol-3-sulphate concentrations (up to 20 ng L⁻¹) were typically 5-fold that of the respective free estrogen concentration in the effluents. This represents a substantial additional ‘potential’ estrogen load arriving in the receiving waters. Estrone-3-glucuronide was found at 9 ng L⁻¹, estradiol-3-glucuronide at 7 ng L⁻¹, and estriol-3-glucuronide at 32 ng L⁻¹ in sewage influent. Except on one occasion, no glucuronide conjugates could be found in the effluent. The results suggest in most cases glucuronide conjugates will be completely transformed in sewage treatment whilst sulphate conjugates will only be partially removed.     

Simultaneous analysis of free and sulfo-conjugated steroid estrogens in artificial urine solution and agricultural soils by high-performance liquid chromatography

A simple and robust analytical method was developed to simultaneously detect and quantify 17β-estradiol (E2), estrone (E1), 17β-estradiol-3-sulphate (E2-3S), and estrone-3-sulphate (E1-3S) in aqueous solutions (calcium chloride and artificial urine solutions) and agricultural soils using high performance liquid chromatography and UV detection. The standards for all four compounds were linear in the range of 0.01 to 1.0 μg mL(-1) (n = 6) and 1.0 to 20 μg mL(-1) (n = 6), respectively, with correlation coefficients > 0.999. The on-column limits of detection at an injection volume of 50 μL and S/N (signal: noise) ratio of 3 were: 9.0 ng mL(-1), 10 ng mL(-1), 5.0 ng mL(-1), and 7.0 ng mL(-1) for E2-3S, E1-3S, E2 and E1, respectively. The limit of detection and quantification in artificial urine solution and CaCl(2) solution was 1.0 ng mL(-1) for all four compounds. Method detection limits for the compounds in the 3 soils ranged from 2 to 2.4 ng g(-1) (E2-3S and E1-3S), and 1.0 to 2.9 ng g(-1) (E2 and E1), respectively.     

Simultaneous analysis of free and sulfo-conjugated steroid estrogens in artificial urine solution and agricultural soils by high-performance liquid chromatography

A simple and robust analytical method was developed to simultaneously detect and quantify 17β-estradiol (E2), estrone (E1), 17β-estradiol-3-sulphate (E2-3S), and estrone-3-sulphate (E1-3S) in aqueous solutions (calcium chloride and artificial urine solutions) and agricultural soils using high performance liquid chromatography and UV detection. The standards for all four compounds were linear in the range of 0.01 to 1.0 μg mL^?1 (n = 6) and 1.0 to 20 μg mL^?1 (n = 6), respectively, with correlation coefficients > 0.999. The on-column limits of detection at an injection volume of 50 μL and S/N (signal: noise) ratio of 3 were: 9.0 ng mL^?1, 10 ng mL^?1, 5.0 ng mL^?1, and 7.0 ng mL^?1 for E2-3S, E1-3S, E2 and E1, respectively. The limit of detection and quantification in artificial urine solution and CaCl₂ solution was 1.0 ng mL^?1 for all four compounds. Method detection limits for the compounds in the 3 soils ranged from 2 to 2.4 ng g^?1 (E2-3S and E1-3S), and 1.0 to 2.9 ng g^?1 (E2 and E1), respectively.     

Environmental transport of endogenous dairy manure estrogens

Although estrogens originating from dairy manure applied to agricultural soils as a fertilizer can potentially contaminate surface water and groundwater, the variables that control transport are poorly understood. Our objective was to assess the potential for off-site movement of endogenous dairy cattle estrogens when manure is applied on fields at agronomically relevant fertilization rates. Estrone (E1), 17α-estradiol (α-E2), and 17β-estradiol (β-E2) were used in laboratory sorption, desorption, and transformation incubations with both manure and an agriculturally relevant soil. Sorption on manure containing 44% organic carbon exceeded sorption on soil containing 0.8% organic carbon by 20 to 150 times, following the pattern of β-E2 > α-E2 > E1. Approximately 20% of E1 and 17% of α-E2 were desorbed from manure, whereas only about 4% of β-E2 was desorbed. Thirty to seventy percent of α-E2 and β-E2 were converted to E1 in soil and manure, making it imperative that transformation reactions be considered when predicting transport and potential biological effects in the environment. Overall results indicate that high organic carbon concentrations and relatively low amounts of desorption inhibit the potential for off-site transport of endogenous dairy manure estrogens.     

Screening of endocrine-disrupting phenols, herbicides, steroid estrogens, and estrogenicity in drinking water from the waterworks of 35 Italian cities and from PET-bottled mineral water

We investigated contamination by endocrine-disrupting chemicals in drinking water from 35 major Italian cities and five popular Italian brands of bottled mineral water. The quality of Italian drinking water was assessed by combing chemical analysis with bioassay to quantify specific estrogenic contaminants and to characterize the actual biological effect of the mixture of chemicals present in drinking water including the contribution of not targeted compounds. The selected contaminants were natural and synthetic steroid estrogens, alkylphenols and bisphenol A, linuron, triazine herbicides, and their metabolites. A specific analytical method was developed based on solid phase extraction of 1 L of water and concentration to 100 μL for quantification by electrospray ionization liquid chromatography tandem mass spectrometry, achieving quantification limits of 0.05–0.36 ng/L for herbicides and 0.64–7.70 ng/L for steroids and phenols. No steroid estrogens were detected in any of the samples, while bisphenol A and nonylphenols were detected in the ranges of 0.82–102.00 and 10.30–84.00 ng/L respectively. Herbicides and their degradation products, when present, were found from slightly above the quantification limits up to 49.91 ng/L, mainly from cities in northern Italy. Chemical analyses were complemented by the performance of a bioassay for the determination of the estrogenic activity in the extracts based on the transactivation of estrogen receptor α-transfected reporter HeLa-ERE-Luciferase-Neomycin cell line. Activity was generally low with maximum estrogenicity of 13.6 pg/L estradiol equivalents.     

β-Estradiol and ethinyl-estradiol contamination in the rivers of the Carpathian Basin

17β-Estradiol (E2) and 17α-ethinyl estradiol (EE2), which are environmental estrogens, have been determined with LC-MS in freshwater. Their sensitive analysis needs derivatization and therefore is very hard to achieve in multiresidue screening. We analyzed samples from all the large and some small rivers (River Danube, Drava, Mur, Sava, Tisza, and Zala) of the Carpathian Basin and from Lake Balaton. Freshwater was extracted on solid phase and derivatized using dansyl chloride. Separation was performed on a Kinetex XB-C18 column. Detection was achieved with a benchtop orbitrap mass spectrometer using targeted MS analysis for quantification. Limits of quantification were 0.05 ng/L (MS1) and 0.1 ng/L (MS/MS) for E2, and 0.001 ng/L (MS1) and 0.2 ng/L (MS/MS) for EE2. River samples contained n.d.–5.2 ng/L E2 and n.d.–0.68 ng/L EE2. Average levels of E2 and EE2 were 0.61 and 0.084 ng/L, respectively, in rivers, water courses, and Lake Balaton together, but not counting city canal water. EE2 was less abundant, but it was still present in almost all of the samples. In beach water samples from Lake Balaton, we measured 0.076–0.233 E2 and n.d.–0.133 EE2. A relative high amount of EE2 was found in river Zala (0.68 ng/L) and in Hévíz-Páhoki canal (0.52 ng/L), which are both in the catchment area of Lake Balaton (Hungary).     

Sorption and Desorption of Three Endocrine Disrupters in Soils

Sorption of the estrogens estrone (E1), 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) on four soils was examined using batch equilibrium experiments with initial estrogen concentrations ranging from 10 to 1000 ng mL^?1. At all concentrations, >85% of the three estrogens sorbed rapidly to a sandy soil. E1 sorbed more strongly to soil than E2 or EE2. Partial oxidation of E2 to E1 was observed in the presence of soils. Autoclaving was more effective at reducing this conversion than inhibition with sodium azide or mercuric chloride, and had little effect on sorption, relative to the chemical microbial inhibitors. Sorption of EE2 was greater for fine-textured than coarse-textured soils, but greater than 90% of EE2 sorbed onto all four soils. The greatest degree of desorption of estrogens from the sandy soil occurred with the lowest initial concentration of 10 ng mL^?1 and reached levels ≥80% for E1 and E2. Desorption of EE2 was greater in coarser textured soils than finer-textured soils. Again, relative desorption from all soils was greatest with low initial concentrations. Therefore, at environmentally relevant concentrations, estrogens quickly sorb to soils, and soils have a large capacity to bind estrogens, but these endocrine-disrupting compounds can become easily desorbed and released into the aqueous phase.     

17 β-estradiol mineralization in human waste products and soil in the presence and the absence of antimicrobials

Natural steroidal estrogens, such as 17 β-estradiol (E2), as well as antimicrobials such as doxycycline and norfloxacin, are excreted by humans and hence detected in sewage sludge and biosolid. The disposal of human waste products on agricultural land results in estrogens and antibiotics being detected as mixtures in soils. The objective of this study was to examine microbial respiration and E2 mineralization in sewage sludge, biosolid, and soil in the presence and the absence of doxycycline and norfloxacin. The antimicrobials were applied to the media either alone or in combination at total rates of 4 and 40 mg kg^?1, with the 4 mg kg^?1 rate being an environmentally relevant concentration. The calculated time that half of the applied E2 was mineralized ranged from 294 to 418 days in sewage sludge, from 721 to 869 days in soil, and from 2,258 to 14,146 days in biosolid. E2 mineralization followed first-order and the presence of antimicrobials had no significant effect on mineralization half-lives, except for some antimicrobial applications to the human waste products. At 189 day, total E2 mineralization was significantly greater in sewage sludge (38 ±0.7%) > soil (23 ±0.7%) > biosolid (3 ±0.7%), while total respiration was significantly greater in biosolid (1,258 mg CO₂) > sewage sludge (253 mg CO₂) ≥ soil (131 mg CO₂). Strong sorption of E2 to the organic fraction in biosolid may have resulted in reduced E2 mineralization despite the high microbial activity in this media. Total E2 mineralization at 189 day was not significantly influenced by the presence of doxycycline and/or norfloxacin in the media. Antimicrobial additions also did not significantly influence total respiration in media, except that total CO₂ respiration at 189 day was significantly greater for biosolid with 40 mg kg^?1 doxycycline added, relative to biosolid without antimicrobials. We conclude that it is unlikely for doxycycline and norfloxacin, or their mixtures, to have a significant effect on E2 mineralization in human waste products and soil. However, the potential for E2 to be persistent in biosolids, with and without the presence of antimicrobials, is posing a challenge for biosolid disposal to agricultural lands.     

A2O工艺中雌激素的行为变化和去除机理

研究了厌氧-缺氧-好氧(A2O)活性污泥工艺对生活污水中天然雌激素雌酮(Estrone,E1)、17β-雌二醇(17β-Estradiol,E2)以及17α-乙炔基雌二醇(17α-Ethynylestradiol,EE2)的去除性能。在对COD、N和P具有良好去除效果的前提下,对E1、E2和EE2的去除率可分别达到92.7%、100%和62.7%。通过对各反应单元内3种雌激素的物料平衡分析,表明A2O工艺对雌激素的去除主要发生在厌氧段和好氧段。以失活污泥作为对照组,好氧硝化过程中雌激素去除的小试实验发现,好氧过程中E1、E2的去除主要依靠生物降解作用,而EE2的去除则主要依赖于活性污泥对其的吸附作用。     

Occurrence,sorption, and transformation of free and conjugated natural steroid estrogens in the environment

Environmental Science and Pollution Research - Natural steroid estrogens (NSEs), including free estrogens (FEs) and conjugated estrogens (CEs), are of emerging concern globally among public and...     

Dissolved organic carbon from sewage sludge and manure can affect estrogen sorption and mineralization in soils

In this study, effects of sewage sludge and manure borne dissolved organic carbon (DOC) on 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) sorption and mineralization processes were investigated in three agricultural soils. Batch equilibrium techniques and equilibrium dialysis methods were used to determine sorption mechanisms between DOC, estrogens and the soil solid phase. It was found that that the presence of organic waste borne DOC decreased estrogen sorption in soils which seems to be controlled by DOC/estrogen complexes in solution and by exchange processes between organic waste derived and soil borne DOC. Incubation studies performed with ¹⁴C-estrogens showed that DOC addition decreased estrogen mineralization, probably due to reduced bioavailability of estrogens associated with DOC. This increased persistence combined with higher mobility could increase the risk of estrogen transport to ground and surface waters.     

Biotransformation of estrogens in nitrifying activated sludge under aerobic and alternating anoxic/aerobic conditions.

Natural and synthetic estrogens present in municipal wastewater can be biodegraded during treatment, particularly in activated sludge. The objective was to assess the extent of transformation of 17-beta-estradiol (E2) and 17-alpha-ethinylestradiol (EE2) by nitrifying activated sludge and evaluate potential relationships between availability of oxygen, nitrification rate, and estrogen removal. For each batch experiment, two reactors were set up--aerobic and alternating anoxic/aerobic-which were then amended with E2 and EE2 from methanolic stock solutions. The EE2 was persistent under anoxic conditions; under aerobic conditions, the observed level of its removal was 22%. The E2 was readily converted to estrone (El)--faster under aerobic (nitrifying) than anoxic (denitrifying) conditions. During the initial anoxic conditions, a metabolite consistent with 17-alpha-estradiol transiently accumulated and was subsequently removed when the reactor was aerated. Higher removal rates of estrogens were associated with higher nitrification rates, which supports the contention that the nitrifying biomass was responsible for their removal.     

A^2O工艺中雌激素的行为变化和去除机理

研究了厌氧-缺氧-好氧（A2O）活性污泥工艺对生活污水中天然雌激素雌酮（Estrone,E1）、17β-雌二醇（17β-Estradiol,E2）以及17α-乙炔基雌二醇（17α-Ethynylestradiol,EE2）的去除性能。在对COD、N和P具有良好去除效果的前提下,对E1、E2和EE2的去除率可分别达到92.7%、100%和62.7%。通过对各反应单元内3种雌激素的物料平衡分析,表明A2O工艺对雌激素的去除主要发生在厌氧段和好氧段。以失活污泥作为对照组,好氧硝化过程中雌激素去除的小试实验发现,好氧过程中E1、E2的去除主要依靠生物降解作用,而EE2的去除则主要依赖于活性污泥对其的吸附作用。     

Sorption and desorption of three endocrine disrupters in soils

Sorption of the estrogens estrone (E1), 17beta-estradiol (E2) and 17alpha-ethynylestradiol (EE2) on four soils was examined using batch equilibrium experiments with initial estrogen concentrations ranging from 10 to 1000 ng mL-1. At all concentrations, >85% of the three estrogens sorbed rapidly to a sandy soil. E1 sorbed more strongly to soil than E2 or EE2. Partial oxidation of E2 to E1 was observed in the presence of soils. Autoclaving was more effective at reducing this conversion than inhibition with sodium azide or mercuric chloride, and had little effect on sorption, relative to the chemical microbial inhibitors. Sorption of EE2 was greater for fine-textured than coarse-textured soils, but greater than 90% of EE2 sorbed onto all four soils. The greatest degree of desorption of estrogens from the sandy soil occurred with the lowest initial concentration of 10 ng mL-1 and reached levels >or=80% for E1 and E2. Desorption of EE2 was greater in coarser textured soils than finer-textured soils. Again, relative desorption from all soils was greatest with low initial concentrations. Therefore, at environmentally relevant concentrations, estrogens quickly sorb to soils, and soils have a large capacity to bind estrogens, but these endocrine-disrupting compounds can become easily desorbed and released into the aqueous phase.     

The effects of soil and Trifolium repens (white clover) on the fate of estrogen

In this study, we investigated the behavior of estrogens in the rhizosphere of white clover (Trifolium repens, clover hereafter) with two different pot tests, using soil and agar as growth media. In a pot test using agar spiked with estrogen, the estrogen concentration in the agar with clover decreased to non-detectable levels within one month, while in the agar without clover, 60% of initially added estrogen remained after one month. The half-lives of estrone (E1) and 17β -estradiol (E2) in the agar with clover were 2.4–3.8 and 13.2 d, respectively. The dissipation of E1 followed first-order rate law, while that of E2 fitted a zero-order reaction, indicating that they had different mechanisms of dissipation from agar. In the soil pot test, the behavior of E1 and E2 was not influenced by clover. An initial rapid decrease in the amount of estrogen extracted by methanol/acetic acid was followed by persistence for 1–3 months, regardless of presence of clover. Moreover, in three weeks E1 and E2 were only partly degraded by microbes extracted from the soil used in the pot test. In this study, abiotic degradation of estrogens and sorption of estrogen to soil, rather than the effects of soil microbes and clover, contributed to the initial rapid dissipation of estrogens in the soil. However, the results of the agar pot test suggested that vegetation such as clover may significantly contribute to removal of estrogens when estrogens in aqueous phase are discharged with surface runoff and preferential flow after heavy rain in agricultural fields, or when present in soils with low estrogen sorptivity.     

Oxidation of natural and synthetic hormones by the horseradish peroxidase enzyme in wastewater

Steroid estrogens, including both natural estrogens (e.g., estrone - E1; 17beta-estradiol - E2; and estriol - E3) and synthetic estrogens (e.g., 17alpha-ethinylestradiol - EE2), are known as endocrine-disrupting compounds. The objective of this research was to evaluate the feasibility of the enzymatic oxidation of estrogens and to optimize this process in municipal wastewater contaminated with steroid estrogens using horseradish peroxidase (HRP) and hydrogen peroxide. An initial HRP activity of 0.02 U ml(-1) was sufficient to completely remove EE2 from the synthetic solution, although greater HRP doses (up to 0.06 U ml(-1)) were required to remove E1, E2 and E3. The optimal molar peroxide-to-substrate ratio was determined to be approximately 0.45. Based on the Michaelis-Menten kinetics, the HRP had an increasing reactivity with E1, E3, E2, and EE2, in increasing order. In real activated sludge process effluent, an HRP dose of 8-10 U ml(-1) was required to completely remove all of the studied estrogens, while only 0.032 U ml(-1) of HRP was necessary to treat synthetic water containing the same estrogen concentrations.