Occurrence of estrogenic effects in sewage and industrial wastewaters in Beijing, China

Estrogenic potencies of the effluents or water samples from wastewater treatment plants (WWTPs), industries and hospitals and some receiving rivers in Beijing city were estimated by using a human estrogen receptor recombinant yeast assay. Estrogenic activity of industrial wastewaters was found to range from 0.1 to 13.3 ng EEQ/L and decreased to the range of 0.03-1.6 ng EEQ/L after treatment. Estrogenic activity in WWTP influent ranged from 0.3 to 1.7 ng EEQ/L and decreased to the range of 0.05-0.5 ng EEQ/L after treatment. In the receiving river waters, the estrogenic effect range was 0.1-4.7 ng EEQ/L. These data suggest that treated industrial effluents and WWTP effluents of concern are not the only source of estrogenic pollution in surface waters in Beijing city. EEQ levels in Beijing river water are likely attributable to untreated municipal and industrial wastewaters discharged directly into the river.     

Biological assessment of bisphenol A degradation in water following direct photolysis and UV advanced oxidation

Endocrine disrupting compounds (EDCs) are exogenous environmental chemicals that can interfere with normal hormone function and present a potential threat to both environmental and human health. The fate, distribution and degradation of EDCs is a subject of considerable investigation. To date, several studies have demonstrated that conventional water treatment processes are ineffective for removal of most EDCs and in some instances produce multiple unknown transformation products. In this study we have investigated the use of direct photolysis with low-pressure (LP) Hg UV lamps and UV+hydrogen peroxide (H(2)O(2)) advanced oxidation process (AOP) for the degradation of a prototypic endocrine disrupter, bisphenol A (BPA), in laboratory water. Removal rates of BPA and formation of degradation products were determined by high performance liquid chromatography (HPLC) analysis. Changes in estrogenic activity were evaluated using both in vitro yeast estrogen screen (YES) and in vivo vitellogenin (VTG) assays with Japanese medaka fish (Oryzias latipes). Our results demonstrate that UV alone did not effectively degrade BPA. However, UV in combination with H(2)O(2) significantly removed BPA parent compound and aqueous estrogenic activity in vitro and in vivo. Removal rates of in vivo estrogenic activity were significantly lower than those observed in vitro, demonstrating differential sensitivities of these bioassays and that certain UV/AOP metabolites may retain estrogenic activity. Furthermore, the UV/H(2)O(2) AOP was effective for reducing larval lethality in treated BPA solutions, suggesting BPA degradation occurred and that the degradation process did not result in the production of acutely toxic intermediates.     

Endocrine disrupting chemicals accumulate in earthworms exposed to sewage effluent

Endocrine disrupting chemicals (EDCs) can alter endocrine function in exposed animals. Such critical effects, combined with the ubiquity of EDCs in sewage effluent and potentially in tapwater, have led to concerns that they could be major physiological disruptors for wildlife and more controversially for humans. Although sewage effluent is known to be a rich source of EDCs, there is as yet no evidence for EDC uptake by invertebrates that live within the sewage treatment system. Here, we describe the use of an extraction method and GC-MS for the first time to determine levels of EDCs (e.g., dibutylphthalate, dioctylphthalate, bisphenol-A and 17beta-estradiol) in tissue samples from earthworms (Eisenia fetida) living in sewage percolating filter beds and garden soil. To the best of our knowledge, this is the first such use of these techniques to determine EDCs in tissue samples in any organism. We found significantly higher concentrations of these chemicals in the animals from sewage percolating filter beds. Our data suggest that earthworms can be used as bioindicators for EDCs in these substrates and that the animals accumulate these compounds to levels well above those reported for waste water. The potential transfer into the terrestrial food chain and effects on wildlife are discussed.     

Distribution of endocrine disruptors in the Llobregat River basin (Catalonia,NE Spain)

The Llobregat basin is a Mediterranean fluvial system with major agricultural, urban and industrial impacts. We combined chemical quantification by liquid chromatography–mass spectrometry with electrospray interface (LC-ESI-MS) and the recombinant yeast assay (RYA) assays to estimate the loads of endocrine disrupting compounds (EDCs) along the basin. Chemical analysis revealed maximum concentrations (at μg l⁻¹ level) of alkylphenols at the lower course of the Llobregat River, which correlated with high levels of estrogenic activity detected by RYA. Analysis by RYA and LC-ESI-MS of influent and effluent waters from four sewage treatment plants (STP) discharging into the basin showed the removal of 80–95% of EDCs by STP treatment. Chemical analysis data and RYA data showed a quasi-linear correlation, demonstrating the complementariness of both methods. Our data suggest that the concentrations of the analysed compounds were enough to explain the total estrogenicity of water and STP samples from the Llobregat basin.     

Distribution of endocrine disruptors in the Llobregat River basin (Catalonia, NE Spain)

The Llobregat basin is a Mediterranean fluvial system with major agricultural, urban and industrial impacts. We combined chemical quantification by liquid chromatography–mass spectrometry with electrospray interface (LC-ESI-MS) and the recombinant yeast assay (RYA) assays to estimate the loads of endocrine disrupting compounds (EDCs) along the basin. Chemical analysis revealed maximum concentrations (at μg l⁻¹ level) of alkylphenols at the lower course of the Llobregat River, which correlated with high levels of estrogenic activity detected by RYA. Analysis by RYA and LC-ESI-MS of influent and effluent waters from four sewage treatment plants (STP) discharging into the basin showed the removal of 80–95% of EDCs by STP treatment. Chemical analysis data and RYA data showed a quasi-linear correlation, demonstrating the complementariness of both methods. Our data suggest that the concentrations of the analysed compounds were enough to explain the total estrogenicity of water and STP samples from the Llobregat basin.     

Estrogenic and dioxin-like potency in each step of a controlled landfill leachate treatment plant in Japan

The estrogenic activity (by E-screen bioassay), the concentrations of PCBs, PCDDs/PCDFs (and their resulting toxicity equivalents, TEQ) and several endocrine disrupting chemicals (EDCs: e.g., bisphenol A, nonylphenol, Butyl benzylpthalate (BBP), di-n-butylphthalate (DBP), 17alpha-ethynyl-estradiol or 4-octylphenol) have been analyzed from leachates of each step (before treatment, after biodegradation/sedimentation and after charcoal treatment) of a controlled landfill leachate treatment plant. The comparison of the effluent of the examined landfill leachate treatment plant with water from a nearby river in this study indicated no additional dioxin-like (e.g., TEQ: 0.027 compared to 1.01 pg TEQ/l; PCBs: 1.2 compared to 3.9 ng/l) or estrogenic impact (2.8 compared to 3.5 ng estradiol equivalents EE/l; analyzed by E-screen bioassay) from the leachate treatment plant into the surrounding water environment. The impact of dioxin-like compounds from uncleaned leachates into the final cleaned effluents could be sufficiently reduced by the leachate treatment plant for PCDDs (75%), PCDFs (62%), dioxin-like PCBs (97%), and the sum of TEQ (78%). The leachate treatment plant also achieved a reduction of the estrogenic activity as determined by E-screen (from 4.8 to 2.8 ng EE/l = 42%), by GC/MS for bisphenol A (>96% and nonylphenol (>98%) or by ELISA for estradiol (>80%). Additionally, for the validation of the E-screen, five known endocrine disrupting chemicals (EDCs: bisphenol A, BBP, DBP, 17 alpha-ethynyl-estradiol, 4-octylphenol) were analyzed. The EC50 values and estradiol equivalents factors (EEFs) for the five EDCs determined in this study were comparable to previously published data. The combined biological and chemical trace analysis data have provided valuable information on the relative contribution of natural, synthetic, and non-steroidal anthropogenic chemicals to the estrogenic and dioxin-like activity in leachates from a wastewater treatment plant, and water from a nearby river.     

Effect directed analysis and mixture effects of estrogenic compounds in a sediment of the river Elbe

INTRODUCTION: Endocrine disrupting chemicals (EDCs) are present in the environment and can have serious effects on humans and wildlife. For the establishment of environmental quality guidelines and regulation of EDCs, a better understanding and knowledge of the occurrence and the behavior of environmental EDCs is necessary. The aim of the present study was to comprehensively identify substances that are responsible for the estrogenic effect of an environmental sediment sample taken from the river Elbe/Germany. DISCUSSION: The estrogenic effect of the organic sediment extract was determined using the yeast-estrogen-screen (YES). The sample was fractionated by liquid chromatography (LC) for effect directed analysis. The composition of estrogen-active fractions was further investigated by gas chromatography-mass spectrometry and high-resolution LC-MS analysis. The composition of the environmental sample was rebuilt with pure compounds in order to assess the partition of estrogenic activity caused by the identified compounds. The organic sediment extract showed an estrogenic potential of 1.9?±?0.4 ng/g ethinylestradiol equivalents in the sediment. The most prominent contaminants with an estrogenic potential were 17β-estradiol, estrone, and 4-iso-nonylphenols, but other xenoestrogens like bisphenol A and stigmasterol could be found as well. A rebuild of the sample was measured in the YES in order to investigate mixture effects. About 67 % of the observed estrogenic effect in the sediment extract could be explained by a mixture which contained all identified compounds. Chlorophene (o-benzyl-p-chlorophenol)-a widely used antiseptic that was also identified in the sediment extract-has xenoestrogenic properties in the YES that are in the range of other xenoestrogens like 4-n-nonylphenol. This is the first report on chlorophene acting as a xenoestrogen.     

Removal of estrone, 17α-ethinylestradiol,and 17ß-estradiol in algae and duckweed-based wastewater treatment systems

Background, aim, and scope  

Many pollutants have received significant attention due to their potential estrogenic effect and are classified as endocrine disrupting compounds (EDCs). Because of possible ecological effects and increased attention for water reuse schemes, it is important to increase our understanding of the EDC removal capacities of various wastewater treatment systems. However, there has so far been little research on the fate and behavior of EDCs in stabilization pond systems for wastewater treatment, which represent an important class of wastewater treatment systems in developing countries because of their cost-effectiveness. The aim of this work is to study the fate and behavior of EDCs in algae and duckweed ponds. Because the synthetic hormone 17α-ethinylestradiol (EE2) and the natural hormones estrone (E1), as well as 17β-estradiol (E2), have been detected in effluents of sewage treatment plants and been suggested as the major compounds responsible for endocrine disruption in domestic sewage; E1, E2, and EE2 were therefore chosen as target chemicals in this current work.     

Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations,sampling season and location

In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between and were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in than in and . Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in . Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to any particular pesticides. Besides, the contribution of non-analyzed xeno-estrogens as alkylphenol(-ethoxylates) and bisphenol-A to the estrogenic potency of rainwater could not be excluded. Further research should focus on the chemical identification of estrogenic compounds in rainwater. In addition, more attention should be given to the ecological consequences of atmospheric deposition of individual pesticides and of total toxic potencies that regularly exceed environmental criteria for Dutch surface waters and/or toxic threshold values for aquatic organisms.     

Determination of estrogenic compounds in wastewater using liquid chromatography-tandem mass spectrometry with electrospray and atmospheric pressure photoionization following desalting extraction

Two complementary LC-MS ionization methods, electrospray (ESI) and atmospheric pressure photoionization (APPI), have been optimized to determine three natural estrogenic compounds (estrone, 17beta-estradiol and estriol) and two synthetic estrogenic compounds (17alpha-ethynylestradiol and diethylstilbestrol) in the influent and effluent of wastewater treatment plants (WWTPs). The wastewater samples were first subjected to solid-phase extraction coupled with desalting extraction to remove matrix interference. The analytes were then detected using liquid chromatography-tandem mass spectrometry (LC-MS-MS) with ESI and dopant-assisted (DA) APPI to evaluate the ion suppression effect and to complement the detection and quantification of estrogenic compounds in complex wastewater samples. The average ion suppression factors for the extracts of the WWTP influent analyzed using ESI and APPI were 52+/-5% and 27+/-7%, respectively. The sensitivity and ionization efficiency of the LC-ESI-MS-MS system decreased dramatically when a complex matrix was present in the WWTP influent sample. Estrogenic compounds could be detected in the WWTP influent and effluent samples at concentrations below the parts-per-billion level. The lower detection limits obtained when using ESI and the higher matrix tolerance of the APPI method allowed the complete quantification of estrogenic compounds in very complex samples in a complementary manner.     

Occurrence, distribution, and seasonal variation of estrogenic compounds and antibiotic residues in Jiulongjiang River, South China

Background and purpose

Estrogenic compounds and antibiotic residues in environment are receiving significant attention because of their potential adverse effects on ecosystems and human health. The objectives of this study were to determine the occurrence and seasonal variability of eight kinds of estrogenic compounds and 14 antibiotics. The study developed an occurrence database of the estrogenic compounds and antibiotics in spatial and temporal scale in Jiulongjiang River, South China, to provide useful information for environmental management of this region.

Methods

Eight estrogenic compounds and 14 antibiotic compounds were detected in Jiulongjiang River from 19 sampling sites during high-flow and low-flow season in surface water. The samples were preconcentrated by solid-phase extraction for analysis. Eight estrogenic compounds were analyzed by gas chromatography mass spectrometry (Agilent 7890A-5975C), and antibiotics were determined by liquid chromatography-tandem mass spectrometry (LC?CMS/MS) system (ABI 3200 Q TRAP).

Results

All target compounds could be detected, except 17??-ethynylestradiol, sulfamerazine, and ofloxacin. The median concentrations for seven estrogenic compounds ranged from 6.00 to 610.72?ng/L, with the detection frequency range of 16.00?C100%. However, the detection frequencies of 13 antibiotics detected varied from 50% to 100%, with the median concentrations ranged from 0.89 to 117.97?ng/L. Seasonal variations were obvious for most estrogenic compounds in Jiulongjiang River, except for octylphenol and estriol. There were significant (P?Conclusion The Jiulongjiang River water was more seriously contaminated by diethylstilbestrol, estrone, sulfamethazine, and tetracyclines. Higher overall concentration levels of estrogenic compounds and antibiotics were detected in low-flow water than those in high-flow water. The pollution of estrogenic compounds and antibiotics in Jiulongjiang River mainly came from municipal sewage and livestock breeding activities.     

Biological response of high-back crucian carp (Carassius auratus) during different life stages to wastewater treatment plant effluent

This study presents the adverse effects of endocrine disrupting chemicals (EDCs) in effluent of wastewater treatment plants (WwTPs) on fish health. A study of chronic exposure to WwTPs effluent for 10 months was undertaken in high-back crucian carp (Carassius auratus) during different life stages, covering early-life-stage (ELS), prespawning period, and postspawning period. Condition factor (CF), gonadosomatic index (GSI), hepatosomatic index (HSI), and plasma vitellogenin (VTG) levels were employed as indicators to assess biological effects of effluent on this gynogenesis species. Meanwhile, some high-back crucian carp were caged in Demonstration Base of Biological Purification for Filter-feeding Fish (hereinafter, Demonstration Base), as WwTPs effluent exposure controls. In the meantime, a depuration study was carried out to determine whether or not the estrogenic effects caused by effluent exposure could be reduced after moving fish into EDCs-free water. CF, HSI, GSI, and plasma Vtg levels of high-back crucian carp caged in Demonstration Base were generally in accordance with seasonal change. Effluent exposure inhibited gonadal growth, reducing GSI in ELS while increasing it around spawning, sharpened liver burdens, increasing HSI, and induced abnormal Vtg expression in juvenile high-back crucian carp, augmenting Vtg concentrations in plasma. Around spawning period, Vtg in high-back crucian carp were mainly induced by endogenous estrogens, and EDCs in effluent had less influence on them. Staying in EDCs-free water for 30 days made high-back crucian carp recover from effects of previous effluent exposure, relieving inhibition of gonadal development and hypertrophy of liver as well as reducing Vtg induced by EDCs in effluent. The results revealed that high-back crucian carp in ELS are more sensitive to WwTPs effluent exposure. Additionally, the depuration study showed a clearance of the estrogenic effects caused by effluent.     

Implications of research on endocrine disruption for the environmental risk assessment, regulation and monitoring of chemicals in the European Union

We assess the implications which research on endocrine disrupting chemicals (EDCs) has for the regulation of synthetic substances and for the protection of the environment, particularly under the forthcoming European Union (EU) REACH legislation. EDCs present regulatory problems inter alia because they can act additively at concentrations which are individually harmless, and they may have non-classical dose (concentration)-response relationships at low exposure levels. Furthermore, current in vivo testing routines were not specifically designed to assess the endocrine disrupting properties of chemicals, whilst in silico and in vitro methods have only limited applicability and availability for this purpose. We need to ensure that the assessment approaches specified in the draft REACH legislation and Technical Guidance are able to evaluate EDCs efficiently. However, it must also be recognised that environmental monitoring procedures in Europe will need to be improved to detect EDCs that have evaded identification, and where appropriate, control, under REACH.     

再生水中内分泌干扰物质的存在及处理现状

随着污水回用大力开展,人们与再生水的接触机会增加,必须保障再生水的水质安全.资料调研发现,作为再生水水源的污水处理厂出水中含有多种内分泌干扰物质,活性炭吸附、超滤、高级氧化技术及膜技术可有效去除再生水中的内分泌干扰物质,但鉴于经济和管理上的困难,目前仍不能全面推广.中国常用的澄清(混凝沉淀)/过滤工艺不能有效去除污水处理厂二级出水中的内分泌十扰物质,这对接触人群的健康产生潜在威胁,对生物种群也有一定的影响.生物处理方法未来有可能成为去除内分泌干扰物质很有前途的方法.     

中国地表饮用水水源地有机类内分泌干扰物污染现况分析

有机类内分泌干扰物(EDCs)是一类对人类和生物内分泌系统产生干扰,并可造成其紊乱的特殊外来物质,对人类和生物健康有极大的危害.以与人类生活密切相关的地表饮用水水源地中的有机类EDCs为研究重点,对中国目前地表饮用水水源地EDCs污染状况、污染物种类及其来源及可能的污染途径进行了评述.目前,中国各地区地表饮用水水源地均有有机类EDCs检出.其中以有机氯农药六六六及其异构体、DDT及其代谢产物和多氯联苯检出率最高,且某些地区检测浓度相当高,主要来源是农药使用和污水排放.国内外对地表饮用水水源地EDCs突发污染事件的研究均较少,应引起高度重视.     

Occurrence, distribution, and sources of six phenolic endocrine disrupting chemicals in the 22 river estuaries around Dianchi Lake in China

The objectives of this study are to track the occurrence, distribution, and sources of phenolic endocrine disrupting compounds (EDCs) in the 22 rivers around Dianchi Lake in China, to estimate the input and output amounts of phenolic EDCs in the water system, and to provide more comprehensive fundamental data for risk assessment and contamination control of phenolic EDCs in aquatic environment. Six phenolic EDCs were systematically evaluated in water and surface sediment in the estuaries of those rivers. The water and sediment samples were preconcentrated by solid-phase extraction system and microwave-assisted extraction system, respectively. Phenolic EDCs were analyzed by GC-MS (Thermo Fisher Scientific, USA) after derivatization. Phenolic EDCs were found ubiquitously in the aquatic environment. The total concentrations ranged from 248 to 4,650 ng/L in water, and 113 to 3,576 ng/g dry weight in surface sediment. The residue amount of phenolic EDCs in Dianchi Lake was 258 kg/a. Concentrations of the phenolic EDCs in the Lake decreased with increase in distance to the estuaries of those rivers which run through urban and industrial areas. The rivers seriously contaminated by phenolic EDCs were Xin River, Yunliang River, Chuanfang River, Cailian River, Jinjia River, Zhengda River, and Daqing River which run through the old area of Kunming City. Satisfying correlations were observed between the concentrations of the target compounds in water and in surface sediment. NP1EO, NP2EO, and BPA were identified as the three predominant phenolic EDCs. There were significant correlations between phenolic EDCs and many basic water quality parameters. Urban and industrial areas are the major contributors for phenolic EDCs, especially in Kunming City. Compositional profiles of phenolic EDCs in surface sediment were similar to those in river water. The concentrations of phenolic EDCs in the rivers located in the northwest part of the valley were very high, and posed a potential risk to aquatic organisms and even human. The concentrations of NP2EO, NP1EO, and BPA were at moderate levels of other areas. The basic water quality parameters (TOC, TN, DO, and pH) play important roles on the distribution, fate, and behavior of phenolic EDCs in the valley.     

Plasma vitellogenin in male teleost fish from 43 rivers worldwide is correlated with upstream human population size

It has been previously demonstrated that vitellogenin (VTG) - a precursor egg yolk protein - is produced in male fish exposed to estrogenic compounds in wastewater treatment plant (WWTP) effluent. However, little attention has been given to examine whether any patterns of male VTG production exists across fish species on a global scale. We hypothesized that a composite measure of human population size over river discharge would best explain variations of protein levels in male fish. We compiled VTG data in 13 fish species from 43 rivers receiving municipal WWTP effluent on 3 continents. We found that human population size explained 28% of the variation in male VTG concentrations, whereas population/flow rate failed to significantly correlate with VTG. We suggest this result may be explained by the low solubility of estrogenic compounds, resulting in localized contamination near WWTP outfalls, rather than dilution by river water.     

Distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals in wild fish species from Dianchi Lake, China

The distribution and bioaccumulation of steroidal and phenolic endocrine disrupting chemicals (EDCs) were studied in various tissues of wild fish species from Dianchi Lake, China. In muscle tissue, 4-tert-octylphenol, 4-cumylphenol, 4-nonlyphenol and bisphenol A were detected in fish from each sampling site, with maximal concentrations of 4.6, 4.4, 18.9 and 83.5 ng/g dry weight (dw), respectively. Steroids (estrone, 17β-estradiol 17α-ethynylestradiol and estriol) were found at lower levels (<11.3 ng/g dw) and less frequently in muscle samples. The highest concentrations of steroids and phenols were found in liver, followed by those in gill and the lowest concentration was found in muscle. The field bioconcentration factors (BCFs) of phenols were calculated in fish species ranged from 18 to 97. Moreover, the measured tissue concentrations were utilized in order to estimate water concentration of steroids (4.4-18.0 ng/L). These results showed that steroidal and phenolic EDCs were likely ubiquitous contaminants in wild fish.     

Occurrence and treatment trials of endocrine disrupting chemicals (EDCs) in wastewaters

This study demonstrates that both synthetic and natural endocrine disrupting chemicals (EDCs) (e.g., bisphenol A, estrone and 17beta-estradiol) were found in the crude wastewaters from two wastewater treatment works (WwTWs). Conventional biological processes can lower EDCs concentrations to several tens to hundreds ngl(-1). Since natural EDCs (e.g., estrone and 17beta-estradiol) have biological activity and adverse impact on the environment at extremely low concentrations (several tens of ngl(-1)), further treatment after conventional biological processes is required. Preliminary trials with ferrate(VI) and electrochemical oxidation process demonstrated that both processes can effectively reduce EDCs to very low levels, ranging between 10 and 100ngl(-1), but the former is more effective than the latter to reduce COD concentration in wastewater for given studying conditions.     

Toxic masking and synergistic modulation of the estrogenic activity of chemical mixtures in a yeast estrogen screen (YES)

Background, aim and scope

Estrogenic and non-estrogenic chemicals typically co-occur in the environment. Interference by non-estrogenic chemicals may confound the assessment of the actual estrogenic activity of complex environmental samples. The aim of the present study was to investigate whether, in which way and how seriously the estrogenic activity of single estrogens and the observed and predicted joint action of estrogenic mixtures is influenced by toxic masking and synergistic modulation caused by non-estrogenic chemical confounders.

Materials and methods

The yeast estrogen screen (YES) was adapted so that toxicity and estrogenicity could be quantified simultaneously in one experimental run. Mercury, two organic solvents (dimethyl sulfoxide (DMSO) and 2,4-dinitroaniline), a surfactant (LAS-12) and the antibiotic cycloheximide were selected as toxic but non-estrogenic test chemicals. The confounding impact of selected concentrations of these toxicants on the estrogenic activity of the hormone 17ß-estradiol was determined by co-incubation experiments. In a second step, the impact of toxic masking and synergistic modulation on the predictability of the joint action of 17ß-estradiol, estrone and estriol mixtures by concentration addition was analysed.

Results

Each of the non-estrogenic chemicals reduced the apparent estrogenicity of both single estrogens and their mixtures if applied at high, toxic concentrations. Besides this common pattern, a highly substance- and concentration-dependent impact of the non-estrogenic toxicants was observable. The activity of 17ß-estradiol was still reduced in the presence of only low or non-toxic concentrations of 2,4-dinitroaniline and cycloheximide, which was not the case for mercury and DMSO. A clear synergistic modulation, i.e. an enhanced estrogenic activity, was induced by the presence of slightly toxic concentrations of LAS-12. The joint estrogenic activity of the mixture of estrogens was affected by toxic masking and synergistic modulation in direct proportion to the single estrogens, which allowed for an adequate adaptation of concentration addition and thus unaffected predictability of the joint estrogenicity in the presence of non-estrogenic confounders.

Discussion

The modified YES proved to be a reliable system for the simultaneous quantification of yeast toxicity and estrogen receptor activation. Experimental results substantiate the available evidence for toxic masking as a relevant phenomenon in estrogenicity assessment of complex environmental samples. Synergistic modulation of estrogenic activity by non-estrogenic confounders might be of lower importance. The concept of concentration addition is discussed as a valuable tool for estrogenicity assessment of complex mixtures, with deviations of the measured joint estrogenicity from predictions indicating the need for refined analyses.

Conclusions

Two major challenges are to be considered simultaneously for a reliable analysis of the estrogenic activity of complex mixtures: the identification of known and suspected estrogenic compounds in the sample as well as the substance- and effect-level-dependent confounding impact of non-estrogenic toxicants.

Recommendations and perspectives

The application of screening assays such as the YES to complex mixtures should be accompanied by measures that safeguard against false negative results which may be caused by non-estrogenic but toxic confounders. Simultaneous assessments of estrogenicity and toxicity are generally advisable.