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.     

Detection of estrogenic activity in municipal wastewater effluent using primary cell cultures from three-spined stickleback and chemical analysis

Environmental estrogens are substances that imitate the effects of endogenous estrogens. Effluents from municipal wastewater treatment plants are known to contain substances with estrogenic activity including steroidal estrogens and xenoestrogens. In the current study, a combination of biological and chemical analysis was applied to determine the estrogenic activity in municipal wastewater effluents in Finland. The male three-spined stickleback (Gasterosteus aculeatus) hepatocyte assay with vitellogenin induction as an endpoint was used for the detection of estrogenic activity in solid phase extracts of wastewater effluents, and 17beta-estradiol (E2) as a positive control. The wastewater extracts and E2 were found to induce vitellogenin production. The extracts were also subjected to chromatographic fractionation and the collected fractions were assayed. The only active fraction was the one in which E2, estrone and ethynylestradiol were eluted. Its activity corresponded to the activity of the original wastewater extract. The LC-MS/MS analyses of the wastewater extracts showed that the concentration of estrone was about 65 ng L(-1), the concentration of E2 was less than 1 ng L(-1), while estriol and 17alpha-ethynylestradiol could not be detected. These findings showed that the activity of the wastewater extracts and the chromatographic fraction was much higher than the activity which could have been expected on the base of the chemical analysis. This strongly indicates that other compounds, possibly acting by additivity or synergism, are playing a major role in the induced vitellogenin production by the hepatocytes.     

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.     

Irrigation of treated wastewater in Braunschweig, Germany: an option to remove pharmaceuticals and musk fragrances

In this study the fate of pharmaceuticals and personal care products which are irrigated on arable land with treated municipal wastewater was investigated. In Braunschweig, Germany, wastewater has been irrigated continuously for more than 45 years. In the winter time only the effluent of the sewage treatment plant (STP) of Braunschweig is used for irrigation, while during summer digested sludge is mixed with the effluent. In the present case study six wells and four lysimeters located in one of the irrigated agricultural fields were monitored with regard to the occurrence of 52 pharmaceuticals and two personal care products (PPCPs; e.g. betablockers, antibiotics, antiphlogistics, carbamazepine, musk fragrances, iodinated contrast media (ICM) and estrogens). No differences in PPCP pollution of the groundwater were found due to irrigation of STP effluents with and without addition of digested sludge, because many polar compounds do not sorb to sludge and lipophilic compounds are not mobile in the soil-aquifer. Most of the selected PPCPs were never detected in any of the lysimeter or groundwater samples, although they were present in the treated wastewater irrigated onto the fields. In the groundwater and lysimeter samples primarily the ICM diatrizoate and iopamidol, the antiepileptic carbamazepine and the antibiotic sulfamethoxazole were detected up to several mugl(-1), while the acidic pharmaceuticals, musk fragrances, estrogens and betablockers were likely sorbed or transformed while passing the top soil layer. Potential estrogenic effects are likely to disappear after irrigation, since the most potent steroid estrogens were not measurable.     

Performance evaluation and application of surface-molecular-imprinted polymer-modified TiO2 nanotubes for the removal of estrogenic chemicals from secondary effluents

The removal of estrogenic chemicals during wastewater reclamation has been a great concern. Current advanced treatment processes are inefficient for the removal of estrogenic chemicals from secondary effluents of municipal wastewater treatment plants (WWTPs) due to the coexistence of other pollutants with less environmental significance which are also removed simultaneously. The search for highly selective and low-cost removal methods is warranted. Therefore, surface-molecular-imprinted polymer-modified TiO₂ nanotube (S-MIP-TiO₂ NT) photocatalysts were fabricated, characterized, and tested for the removal of estrogenic pollutants from wastewater in this study for the first time. Scanning electron microscopy and Fourier-transform infrared spectroscopy studies showed that the TiO₂ NTs (with an average diameter of 60 nm) were successfully imprinted with functional groups (i.e., carboxyl). The adsorption selectivity and photocatalytic activity of the S-MIP-TiO₂ NTs towards template compound (17β-estradiol, E2) were improved, compared with neat TiO₂ NTs. Interestingly, S-MIP-TiO₂ NTs exhibited higher adsorption intensity and photocatalytic selectivity at low concentrations (from 10 ng/L to 100 μg/L, as normal estrogenic chemical concentrations in secondary effluents) of E2 than that at high concentrations (from 10 to 1,000 mg/L). It was also found that some representative estrogenic chemicals and estrogenic activity could be selectively and rapidly removed from secondary effluents of municipal wastewater treatment plants using S-MIP-TiO₂ NTs as photocatalysts. In addition, S-MIP-TiO₂ NT photocatalysts exhibited excellent regeneration characteristics. Photocatalytic treatment using S-MIP-TiO₂ NTs could be a promising approach for the effective removal of estrogenic chemicals from secondary effluents of municipal WWTPs.     

Europe-wide survey of estrogenicity in wastewater treatment plant effluents: the need for the effect-based monitoring

A pan-European monitoring campaign of the wastewater treatment plant (WWTP) effluents was conducted to obtain a concise picture on a broad range of pollutants including estrogenic compounds. Snapshot samples from 75 WWTP effluents were collected and analysed for concentrations of 150 polar organic and 20 inorganic compounds as well as estrogenicity using the MVLN reporter gene assay. The effect-based assessment determined estrogenicity in 27 of 75 samples tested with the concentrations ranging from 0.53 to 17.9 ng/L of 17-beta-estradiol equivalents (EEQ). Approximately one third of municipal WWTP effluents contained EEQ greater than 0.5 ng/L EEQ, which confirmed the importance of cities as the major contamination source. Beside municipal WWTPs, some treated industrial wastewaters also exhibited detectable EEQ, indicating the importance to investigate phytoestrogens released from plant processing factories. No steroid estrogens were detected in any of the samples by instrumental methods above their limits of quantification of 10 ng/L, and none of the other analysed classes of chemicals showed correlation with detected EEQs. The study demonstrates the need of effect-based monitoring to assess certain classes of contaminants such as estrogens, which are known to occur at low concentrations being of serious toxicological concern for aquatic biota.     

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.     

Removal of estrogenic activity of natural steroidal hormone estrone by ligninolytic enzymes from white rot fungi

Natural steroidal hormone estrone (E1) was treated with the white rot fungus Phanerochaete sordida YK-624 under ligninolytic condition with low-nitrogen and high-carbon culture medium. E1 decreased by 98% after 5 d of treatment and the activities of ligninolytic enzymes, manganese peroxidase (MnP) and laccase, were detected during treatment, which suggested that the disappearance of E1 is related to ligninolytic enzymes produced extracellularly by white rot fungus. Therefore, E1 was treated with MnP and laccase prepared from the culture of white rot fungi. HPLC analysis demonstrated that E1 disappeared completely in the reaction mixture after 1 h of treatment with either MnP or laccase. Using the yeast two-hybrid assay system, it was also confirmed that both enzymatic treatments completely removed the estrogenic activity of E1 after 2 h. These results strongly suggest that ligninolytic enzymes are effective in removing the estrogenic activity of E1.     

Effects-directed analysis of organic toxicants in wastewater effluent from Zagreb, Croatia

The organic toxicants present in the effluent of the main sewer of the city of Zagreb, Croatia were isolated and identified through the use of effects-directed characterisation techniques. At the time of investigation, the wastewater effluent received no treatment and was comprised of a mixture of effluent from domestic and industrial sources. The organic load of the wastewater was isolated by solid phase extraction and toxicity profiles obtained using reverse-phase HPLC. All procedures were evaluated through the analysis of a series of reference compounds of widely differing polarity. Toxicity profiles for EROD activity (CYP1A induction), vitellogenin induction (estrogenic activity), cytotoxicity (membrane stability and metabolic inhibition) were obtained using a rainbow trout (Oncorhynchus mykiss) primary hepatocyte bioassay. The suite of bioassays showed biological responses after exposure to the raw extracts for all the endpoints tested. However, a combination of mixture toxicity and cytotoxicity in the complex raw extract had some masking effect on the sub-lethal responses of vitellogenin and EROD induction. Bioassay testing of the fine fractions obtained by HPLC produced a range of endpoint-specific toxicity profiles for each sample. A number of compounds were identified by the use of GC-MS and LC-MS/MS as responsible for the observed effects. The steroid estrogens 17 beta-estradiol and estriol were identified by LC-MS/MS as estrogen receptor agonists in two of the estrogenic fractions. In addition, GC-MS analysis identified different alkylphenols, benzophenone and methylparaben which also contributed to the estrogenic activity of the sample. Polycyclic aromatic hydrocarbons (PAHs), alkyl substituted PAHs, nitro-polycyclic aromatic compounds (nitro-PACs), carbazoles and alkyl substituted carbazoles and other known CYP1A inducers were identified by GC-MS analysis as responsible for some of the observed EROD activity. Some active compounds remain unidentified.     

Quantification of steroid sex hormones using solid-phase extraction followed by liquid chromatography-mass spectrometry.

In this study, the occurrence of trace amounts of natural and synthetic steroid estrogens in the aquatic environment was studied using liquid chromatography coupled with electrospray mass spectrometry, following solid-phase extraction (SPE). The SPE was performed with C18 and NH2 cartridges. The first objective was to develop a reliable method for analyzing steroid estrogens (resulting from human and animal excretions) in different matrices. The method developed was then applied to quantify the occurrence of natural and synthetic hormones (estrone [E1], 17beta-estradiol [betaE2], 17alpha-estradiol [alphaE2], estriol [E3], and 17alpha-ethinylestradiol [EE2]) in environmental samples in surface water and wastewater treatment plant (WWTP) influent and effluent. In the WWTP influents, betaE2, alphaE2, and E3 were identified as ranging up to 72.6 ng/L in WWTP influent and to 16 ng/L in WWTP effluent. Analysis o f surface wa ter sampled upstream from the WWTP revealed the presence of all five estrogens, at levels up to 19.8 ng/L. These concentrations of estrogens pose an issue for large and small communities, because they are higher than the recommended guidelines for estrogen-active compounds and because a lot of communities use surface water as drinking-water sources.     

Occurrence, fate and antibiotic resistance of fluoroquinolone antibacterials in hospital wastewaters in Hanoi, Vietnam

Occurrence and behavior of fluoroquinolone antibacterial agents (FQs) were investigated in hospital wastewaters in Hanoi, Vietnam. Hospital wastewater in Hanoi is usually not treated and this untreated wastewater is directly discharged into one of the wastewater channels of the city and eventually reaches the ambient aquatic environment. The concentrations of the FQs, ciprofloxacin (CIP) and norfloxacin (NOR) in six hospital wastewaters ranged from 1.1 to 44 and from 0.9 to 17 micrgl(-1), respectively. Total FQ loads to the city sewage system varied from 0.3 to 14 g d(-1). Additionally, the mass flows of CIP and NOR were investigated in the aqueous compartment in a small wastewater treatment facility of one hospital. The results showed that the FQ removal from the wastewater stream was between 80 and 85%, probably due to sorption on sewage sludge. Simultaneously, the numbers of Escherichia coli (E. coli) were measured and their resistance against CIP and NOR was evaluated by determining the minimum inhibitory concentration. Biological treatment lead to a 100-fold reduction in the number of E. coli but still more than a thousand E. coli colonies per 100ml of wastewater effluent reached the receiving water. The highest resistance was found in E. coli strains of raw wastewater and the lowest in isolates of treated wastewater effluent. Thus, wastewater treatment is an efficient barrier to decrease the residual FQ levels and the number of resistant bacteria entering ambient waters. Due to the lack of municipal wastewater treatment plants, the onsite treatment of hospital wastewater before discharging into municipal sewers should be considered as a viable option and consequently implemented.     

Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes

Bisphenol A (BPA) and nonylphenol (NP) were treated with manganese peroxidase (MnP) and laccase prepared from the culture of lignin-degrading fungi. Laccase in the presence of 1-hydroxybenzotriazole (HBT), the so-called laccase-mediator system, was also applied to remove the estrogenic activity. Both chemicals disappeared in the reaction mixture within a 1-h treatment with MnP but the estrogenic activities of BPA and NP still remained 40% and 60% in the reaction mixtures after a 1-h and a 3-h treatment, respectively. Extension of the treatment time to 12 h completed the removal of estrogenic activities of BPA and NP. The laccase has less ability to remove these activities than MnP, but the laccase-HBT system was able to remove the activities in 6 h. A gel permeation chromatography (GPC) analysis revealed that main reaction products of BPA and NP may be oligomers formed by the action of enzymes. Enzymatic treatments extended to 48 h did not regenerate the estrogenic activities, suggesting that the ligninolytic enzymes are effective for the removal of the estrogenic activities of BPA and NP.     

Overcoming the toxicity effects of municipal wastewater sludge and biosolid extracts in the Yeast Estrogen Screen (YES) assay

For nearly two decades, the Yeast Estrogen Screen (YES) has been used as a valuable tool for determining the total estrogenic potency of various environmental samples, including influent and effluent streams at municipal wastewater plants. However, applying the YES assay to wastewater sludges and stabilized biosolids has been problematic. This is due to co-extracted compounds from the solids either proving toxic to the yeast or masking the presence of estrogenic substances. The present research describes the development and validation of sample preparation steps that mitigate the toxicity effects of municipal wastewater sludge and biosolid samples in the YES assay, while allowing for reliable dose-dependent expression of estrogenic activity. A copper work-up for sulfur removal and chromatographic cleanup with silica and alumina were required in addition to solid-phase extraction to adequately remove interfering compounds. Sample stabilization methods such as autoclaving, lyophilization and formaldehyde treatment were found to be detrimental to the assay. Hence, heat-drying is recommended to prevent cytotoxicity and the degradation of estrogenic substances.     

Free synthetic and natural estrogen hormones in influent and effluent of three municipal wastewater treatment plants.

Three municipal wastewater treatment plants (WWTPs) in southeastern Pennsylvania were sampled to determine the presence and concentrations of 12 natural and synthetic estrogen hormones in the wastewater influent and effluent. The target estrogens were 17alpha-estradiol, estrone, estriol, equilin, 17alpha-dihydroequilin, 17beta-estradiol, 17alpha-ethinyl estradiol, gestodene, norgestrel, levonorgestrel, medrogestone, and trimegestone. One WWTP uses a biofilm reactor (packed-bed trickling filter),and the other two use suspended-growth media (continuously stirred activated sludge reactor and sequential batch reactor). Estrone was detected in all the three plants; estriol and estradiol were detected at two WWTPs; and 17 alpha-dihydroequilin and 17 alpha-ethinyl estradiol were detected at one WWTP. The concentration of estrogens in the influent and effluent of the three treatment plants ranged from 1.2 to 259 ng/L and 0.5 to 49 ng/L, respectively. The percentage removal of estrogens from the aqueous phase ranged from 41 to 99%, except in the case of 17alpha-dihydroequilin; the removal of 17alpha-dihydroequilin was negligible. The suspended-growth media systems showed higher removal efficiencies for estrogens than the biofilm system. The analytical method uses a Varian C-18 solid-phase extraction (Varian Inc., Palo Alto, California), followed by a derivatization with bis(trimethylsilyl)trifluoroacetamide. The detection limits for the estrogen compounds ranged from 0.1 to 10 ng/L using a sample size of 1 L. The method recoveries ranged from 71 to 120%, and the relative standard deviation ranged from 6 to 14% for all the hormones.     

The fate of estrogenic hormones in an engineered treatment wetland with dense macrophytes.

Recently, the estrogenic hormones 17beta-estradiol (E2) and 17alpha-ethinyl estradiol (EE2) have been detected in municipal wastewater effluent and surface waters at concentrations sufficient to cause feminization of male fish. To evaluate the fate of steroid hormones in an engineered treatment wetland, lithium chloride, E2, and EE2 were added to a treatment wetland test cell. Comparison of hormone and tracer data indicated that 36% of the E2 and 41% of the EE2 were removed during the cell's 84-h hydraulic retention time (HRT). The observed attenuation was most likely the result of sorption to hydrophobic surfaces in the wetland coupled with biotransformation. Sorption was indicated by the retardation of the hormones relative to the conservative tracer. Biotransformation was indicated by elevated concentrations of the E2 metabolite, estrone. It may be possible to improve the removal efficiency by increasing the HRT or the density of plant materials.     

Removal of selected natural and synthetic estrogenic compounds in a Canadian full-scale municipal wastewater treatment plant.

The effect of a full-scale municipal wastewater treatment plant (WWTP) and each of the treatment units within the stream on the removal of endocrine-disrupting compounds was evaluated by tracking 17-beta-estradiol (E2), estrone (E1), and 17-alpha-ethinylestradiol (EE2). The overall performance of the WWTP compared well with other plants, as 90.5% removal of E1+E2 and 74.9% removal of EE2 were observed. A larger fraction of EE2 entered the plant in particulate form than E1 and E2, while a lower fraction of EE2 left the plant in particulate form than soluble form. The activated sludge units reduced the concentration of E1+E2 and EE2 in the liquid phase by 88.2% and 44.6%, respectively. The UV treatment process did not reduce the amount of estrogens. The aqueous phase of the tertiary lagoon solids contained higher levels of estrogens compared with the lagoon influent.     

Effects of waterborne exposure to 4-nonylphenol on plasma sex steroid and vitellogenin concentrations in sexually mature male carp (Cyprinus carpio)

4-Nonylphenol (NP) has been shown to elicit estrogenic responses both in vivo and in vitro. The mechanism by which NP exerts estrogenic and other endocrine-modulating effects in vivo remains unclear, however. The goal of this study was to evaluate the ability of NP to elicit estrogenic responses through indirect mechanisms of action involving the modulation of endogenous steroid hormone concentrations. Sexually mature male common carp (Cyprinus carpio) were exposed to aqueous NP concentrations ranging from <0.05 to 5.4 microg NP/l for 28-31 d. Approximately 0.5-3.5 ppm of NP was detected in pooled plasma samples or tissue samples from the carp studied. NP exposure did not significantly increase plasma concentrations of 17beta-estradiol (E2), testosterone (T) or vitellogenin (VTG). Excluding outliers, plasma E2 concentrations ranged from <175 to 700 pg E2/ml. T concentrations ranged from 940 to 24,700 pg T/ml plasma. The greatest VTG concentration detected was 52 microg/ml. One-third of the plasma samples tested contained <1 microg VTG/ml. Overall, the results of this study did not support the hypothesis that exposure to waterborne NP can modulate concentrations of steroid hormones in the plasma of sexually mature male carp. The results did, however, raise a number of questions regarding the utility of estradiol equivalent (EEQ) estimates as a means of predicting in vivo effects of estrogenic substances. Furthermore, they provide information regarding the concentrations and variability of E2, T, and VTG in the plasma of sexually mature male carp, which may aid in design and interpretation of future studies.     

Assessment of fates of estrogens in wastewater and sludge from various types of wastewater treatment plants

We measured five estrogens in the wastewater samples from the municipal wastewater treatment plants (M-WWTPs), livestock wastewater treatment plants (L-WWTPs), hospital WWTPs (H-WWTPs) and pharmaceutical manufacture WWTPs (P-WWTPs) in Korea. The L-WWTPs showed the highest total concentration (0.195-10.4 μg L⁻¹) of estrogens in the influents, followed by the M-WWTPs (0.028-1.15 μg L⁻¹), H-WWTPs (0.068-0.130 μg L⁻¹) and P-WWTPs (0.015-0.070 μg L⁻¹). Like the influents, the L-WWTPs (0.003-0.729 μg L⁻¹) and the M-WWTPs (0.001-0.299 μg L⁻¹) also showed higher total concentration of estrogens in the effluents than the H-WWTPs (0.002-0.021 μg L⁻¹) and P-WWTPs (0.011 μg L⁻¹ in one sample). The L-WWTPs (37.5-543 μg kg⁻¹, dry weight) showed higher total concentrations in sludge than the M-WWTPs (3.16-444 μg kg⁻¹, dry weight) like the wastewater. The distribution of estrogens in the WWTPs may be affected by their metabolism in the human body, their transition through biological treatment processes, and their usage for livestock growth. Unlike the concentration results, the daily loads of estrogens from the M-WWTPs were the highest, which is related to the high capacities of WWTPs.