An overall risk probability-based method for quantification of synergistic and antagonistic effects in health risk assessment for mixtures: theoretical concepts

Purpose

In the assessment of health risks of environmental pollutants, the method of dose addition and the method of independent action are used to assess mixture effects when no synergistic and/or antagonistic effects are present. Currently, no method exists to quantify synergistic and/or antagonistic effects for mixtures. The purpose of this paper is to develop the theoretical concepts of an overall risk probability (ORP)-based method to quantify the synergistic and antagonistic effects in health risk assessment for mixtures.

Method

The ORP for health effects of environmental chemicals was determined from the cumulative probabilities of exposure and effects. This method was used to calculate the ORP for independent mixtures and for mixtures with synergistic and antagonistic effects.

Results

For the independent mixtures, a mixture ORP can be calculated from the product of the ORPs of individual components. For systems of interacting mixtures, a synergistic coefficient and an antagonistic coefficient were defined respectively to quantify the ORPs of each individual component in the mixture. The component ORPs with synergistic and/or antagonistic effects were then used to calculate the total ORP for the mixture.

Conclusions

An ORP-based method was developed to quantify synergistic and antagonistic effects in health risk assessment for mixtures. This represents a first method to generally quantify mixture effects of interacting toxicants.     

Analysis of estrogenic activity in coastal surface waters of the Baltic Sea using the yeast estrogen screen

In the present study, the yeast estrogen screen (YES) has been used to assess the estrogenic activity in surface waters of a coastal region in the German Baltic Sea. Solid-phase extraction using the copolymer Oasis HLB followed by a clean-up on silica was carried out on approximately 50-l water samples. From the final 400 μl extract volume, 100 μl aliquots were used for the measurement of estrogenic activity and for chemical analysis, which was performed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). From 29 samples taken during two campaigns (2003 and 2004) at five different stations 27 samples showed an estrogenic response higher than 10%. The response in the YES was expressed as measured estradiol equivalents (EEQs), which were in the range of 0.01 (Darss Peninsula) to 0.82 ng/l (Inner Wismar Bay). Samples from stations located in inner coastal waters showed higher estrogenic activities than those from outer located stations. A comparison of measured estrogenicity (YES) and calculated estrogenicity (chemical analysis) showed significant differences, probably due to the presence of anti-estrogenic compounds and/or the estrogenic activity of unknown, not identified contaminants. The main contributors to the overall estrogenic activity were synthetic and natural hormones.     

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.     

Influence factors of multicomponent mixtures containing reactive chemicals and their joint effects

Organic chemicals usually coexist as a mixture in the environment, and the mixture toxicity of organic chemicals has received increased attention. However, research regarding the joint effects of reactive chemicals is lacking. In this study, we examined two kinds of reactive chemicals, cyanogenic toxicants and aldehydes and determined their joint effects on Photobacterium phosphoreum. Three factors were found to influence the joint effects of multicomponent mixtures containing reactive chemicals, including the number of components, the dominating components and the toxic ratios. With an increased number of components, the synergistic or antagonistic effects (interactions) will weaken to the additive effects (non-interactions) if the added component cannot yield a much stronger joint effect with an existing component. Contrarily, the joint effect of the mixture may become stronger instead of weaker if the added components can yield a much stronger joint effect than the existing joint effect of the multicomponent mixture. The components that yield the strongest interactions in their binary mixture can be considered the dominating components. These components contribute more to the interactions of multicomponent mixtures than other components. Moreover, the toxic ratios also influence the joint effects of the mixtures. This study provides an insight into what are the main factors and how they influence the joint effects of multicomponent mixtures containing reactive chemicals, and thus, the findings are beneficial to the study of mixture toxicology.     

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.     

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.     

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.     

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.     

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.     

Insights into the structural and conformational requirements of polybrominated diphenyl ethers and metabolites as potential estrogens based on molecular docking

PBDEs and their metabolites are of concern due to their increasing concentrations in the environment and their toxic effects. Knowledge about the toxicological mechanisms of PBDEs and metabolites is urgently needed for further screening. The objective of the present study was to explore the structural and conformational requirements of PBDE compounds as human estrogen receptor alpha (hERα) agonists, and further screened out hERα agonists from PBDE compounds. Molecular docking and postdocking analysis were adopted to attain the aim. The obtained results revealed that PBDEs can be primarily screened for their estrogenicity using score values, hydrogen bonds interaction with amino acid residues Glu353 and/or Arg394 might be important for HO-PBDEs’ estrogenicity. For most MeO-PBDEs, hydrophobic interaction might be the key factor affecting their estrogenic activity. The current study suggested that molecular docking and postdocking analysis can serve as an efficient pre-screening technique for identifying potential estrogens.     

Determination of estrogenic activity by LYES-assay (yeast estrogen screen-assay assisted by enzymatic digestion with lyticase)

In order to enhance the sensitivity and the speed of the yeast estrogen screen (YES)-assay, which has been established in many laboratories for the determination of estrogenic activity of compounds and environmental samples, the LYES-assay, a modified version of the YES-assay including a digestion step with the enzyme lyticase, was developed. With the LYES-assay the estrogenic activities of natural (17β-estradiol E2 and estrone), synthetic (17-ethinylestradiol EE2) and pharmaceutical estrogens (diethylstilbestrol DES) as well as xenoestrogens (4-nonylphenol NP and five parabens) were determined and compared with the results obtained by other in vitro-assays namely the conventional YES-assay, the E-Screen-assay (MCF-7 breast tumor cell proliferation) and a receptor binding-assay (RB) with human estrogen receptors hER- and hER-β. In the case of E2 the LYES-assay had a significantly lower limit of quantification (LOQ) than the conventional YES-assay and even two orders of magnitude lower than the RB-assay. Compared to the E-Screen-assay the LOQ of the LYES-assay was almost one order of magnitude higher. The time required to perform the LYES-assay was as little as seven hours compared to three to five days for the conventional YES-assay. Thus, the LYES-assay is a very good alternative to existing estrogenic in vitro-assays, since it has a good sensitivity, is cheap and much faster than the other assays.     

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.     

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.     

Acute and chronic toxicity of benzotriazoles to aquatic organisms

Purpose

Resulting from their intensive use as corrosion inhibitors in aircraft deicing and anti-icing fluids (ADAF) and for silver protection in dishwasher detergents benzotriazoles (BTs) are widespread in European surface waters. The current study aimed on an ecotoxicological characterization of 1H-benzotriazole (1H-BT) and 5-methyl-1H-benzotriazole (5MBT).

Methods

Acute and chronic OECD guideline tests were conducted with primary producers (Desmodesmus subspicatus, Lemna minor) and two daphnia species (Daphnia magna, Daphnia galeata) to characterize the hazard of these chemicals. Additionally, the estrogenic activity of both BTs was analyzed in vitro using a recombinant yeast estrogen screen (YES).

Results

Both BTs revealed significant effects in acute and chronic experiments, but exhibited no estrogenic activity in the YES. The algal growth test displayed an inhibited cell number increase with effect concentration (EC) values of EC₁₀ 1.18 and 2.86?mg?l^-1 for 1H-BT and 5MBT, respectively. In the Lemna test, EC₁₀ values were 3.94?mg?l^-1 (1H-BT) and 2.11?mg?l^-1 (5MBT). D. magna was also affected with EC₅₀ (48?h) values of 107?mg?l^-1 for 1H-BT and 51.6?mg?l^-1 for 5MBT. D. galeata was more sensitive with an EC₅₀ (48?h) of 14.7?mg 1H-BT l^-1 and 8.13?mg 5MBT l^-1. In the 21-day reproduction tests with D. magna, the EC₁₀ for 5MBT was 5.93?mg?l^-1 while 1H-BT showed no adverse effects. D. galeata turned out to be more sensitive in the chronic study with EC₁₀ values of 0.97?mg?l^-1 for 1H-BT and 0.40?mg?l^-1 for 5 MBT.

Conclusion

Because BTs are regularly found in the aquatic environment at lower ??g l^-1 concentrations reflecting their persistence and poor elimination during wastewater treatment processes, a preliminary risk assessment was conducted. There is little indication that BTs pose a risk for aquatic ecosystems at current exposure levels during most of the year. However, it cannot be excluded that in winter with a higher usage of ADAFs environmental concentrations may well exceed the level that is considered safe for aquatic organisms.     

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.     

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).     

AMEG: the new SETAC advisory group on aquatic macrophyte ecotoxicology

Introduction and background

Primary producers play critical structural and functional roles in aquatic ecosystems; therefore, it is imperative that the potential risks of toxicants to aquatic plants are adequately assessed in the risk assessment of chemicals. The standard required macrophyte test species is the floating (non-sediment-rooted) duckweed Lemna spp. This macrophyte species might not be representative of all floating, rooted, emergent, and submerged macrophyte species because of differences in the duration and mode of exposure; sensitivity to the specific toxic mode of action of the chemical; and species-specific traits (e.g., duckweed's very short generation time).

Discussion and perspectives

These topics were addressed during the workshop entitled “Aquatic Macrophyte Risk Assessment for Pesticides” (AMRAP) where a risk assessment scheme for aquatic macrophytes was proposed. Four working groups evolved from this workshop and were charged with the task of developing Tier 1 and higher-tier aquatic macrophyte risk assessment procedures. Subsequently, a SETAC Advisory Group, the Macrophyte Ecotoxicology Group (AMEG) was formed as an umbrella organization for various macrophyte working groups. The purpose of AMEG is to provide scientifically based guidance in all aspects of aquatic macrophyte testing in the laboratory and field, including prospective as well as retrospective risk assessments for chemicals. As AMEG expands, it will begin to address new topics including bioremediation and sustainable management of aquatic macrophytes in the context of ecosystem services.     

Removal of estrogenic activity of natural and synthetic hormones from a municipal wastewater: efficiency of horseradish peroxidase and laccase from Trametes versicolor

Some researches studied the removal of steroid estrogens by enzymatic treatment, however none verified the residual estrogenicity after the enzymatic treatment at environmental conditions. In this study, the residual estrogenic activities of the key natural and synthetic steroid estrogens were investigated following enzymatic treatment with horseradish peroxidase (HRP) and laccase from Trametes versicolor. Synthetic water and municipal wastewater containing environmental concentrations of estrone, 17beta-estradiol, estriol, and 17alpha-ethinylestradiol were treated. Liquid chromatography-mass spectrometry analysis demonstrated that the studied steroid estrogens were completely oxidized in the wastewater reaction mixture after a 1-h treatment with either HRP (8-10 U ml(-1)) or laccase (20 U ml(-1)). Using the recombinant yeast assay, it was also confirmed that both enzymatic treatments were very efficient in removing the estrogenic activity of the studied steroid estrogens. The laccase-catalyzed process seemed to present great advantages over the HRP-catalyzed system for up-scale applications for the treatment of municipal wastewater.