Development Of A Chemical Analysis Protocol And Application With Whole Estrogenic And Androgenic Assays To Assess Endocrine Disruptor Activity During Wastewater And Sludge Treatment Processes

Endocrine Disrupting Chemicals (EDCs) have been shown to produce changes in the endocrine system of organisms that lead to increases in cancers and abnormalities in reproductive structure and function. Recent research has highlighted the existence of hormonally active compounds in sewage and industrial effluents and their potential for recycling back into the environment - including drinking water supplies- through point sources and non-point sources. Endocrine Disrupters in Wastewater and Sludge Treatment Processes presents the latest research on EDCs, covering the sources, fate, and transport of EDCs in sewage and industrial effluents, and sludge treatment and disposal options in light of effects on receiving environments. In addition, the authors review current legislation, future research needs, and potential management strategies for endocrine disrupters in the environment.

During the past decade, estrogenic contaminants and polybrominated diphenyl ethers (PBDEs) received more and more attention due to their adverse effects as endocrine disruptors. There is a need to examine fate of these contaminants during wastewater treatment and effluent polishing process, as well as during the land application of biosolids as soil amendments, within the context of potable water reuse and sludge application, which have all been widely practiced. Two major research goals guided this research. The first goal was to develop experimental protocols measuring estrogenic activity (including nonylphenol) and PBDEs in environmental samples, especially in organic rich solid samples such as sludges, sediments and soils which are impacted by wastewater and/or application of biosolids. The second objective was to evaluate fate of estrogenic activity and PBDEs during conventional wastewater treatment, effluent polishing, and sludge handling processes including digestion, dewatering, composting, and land application of biosolids by using the protocols developed. The protocol developed to measure estrogenic activity or PBDEs in the solids includes extraction, cleanup, and determination steps. Each step is critical for the successful determination; however cleanup step was the most difficult. In this study, a C18 resin was used as the media to remove the bulk organic interferences in the measurement of estrogenic activity and nonylphenol. In comparison, Florisil was used in the cleanup step for PBDE analysis. In the development of each protocol, mobile phase was carefully selected and optimum cleanup strategy was determined, recovery of analytes during cleanup operation was measured. During the development of method measuring the estrogenic activity, effects of extraction variables such as solvent, pressure, and time were investigated. The performance of each protocol was examined by spike andrecovery experiments. Experiments indicated that estrogenic activity and nonylphenol were largely removed during traditional wastewater treatment, soil aquifer treatment, and surface transport along a wastewater dependent stream. Examination of estrogenic activity and nonylphenol in sludge, sediments in contact with wastewater and mass balance analysis of these estrogenic contaminants in traditional wastewater treatment plants and infiltration basins indicated that both adsorption and biodegradation play important roles. In comparison, estrogenic activity and nonylphenol were persistent during anaerobic sludge digestion. More experiments are warranted to understand fate of PBDEs during sludge digestion process, although limited data show possible degradation.

With its 104 chapters, this Encyclopedia of aquatic ecotoxicology reveals the diversity of issues, problems and challenges that have faced, and are facing today, receiving environments. It also indicates ways by which tools, strategies and future investigations can contribute to correct, minimize, solve and prevent water quality degradation. Structured homogeneously, the chapters convey salient information on historical background, features, characteristics, uses and/or applications of treated topics, often complemented by illustrations and case studies, as well as by conclusions and prospects. This work is most suitable for teaching purposes. Academics, for example, could literally deliver comprehensive lectures to students simply based on chapter outlines and contents. Meet the Authors of the Encyclopedia! Check out 'Meet the Authors' under ADDITIONAL INFORMATION (Right menu).

Endocrine disrupting chemicals (EDCs) are compounds found in the environment that have the potential to disrupt normal endocrine function. Estrogenic EDCs (e-EDCs) is a subclass of EDCs and is defined as substances contaminating the environment that may mimic or inhibit the effect of endogenous estrogen and therefore may influence developmental and reproductive health in humans and animals. The aim of this study was to develop, validate and implement a battery of in vitro and in vivo screening assays for e-EDCs. The study was concluded by implementing this battery of assays to assess the Eerste River, South Africa at three sampling sites, namely Jonkershoek, Stellenbosch sewage treatment works (STW) effluent and Spier for e-EDCs. The control site, Jonkershoek contained very low levels of estrone. Water from this site showed no estrogenic activity when the E-screen and the ER_ induction in MCF-7 cells. Some of the water samples collected at this site tested positive for estrogenicity when analysed with the juvenile tilapia VTG assay, whereas the rest were negative. The estrone levels in the sewage effluent extracts as well as Spier were significantly higher. The assay using ER_ protein induction by the MCF-7 cell line, the MCF-7 proliferation assay and the tilapia in vivo screen for estrogenicity showed that these samples are estrogenic. Results obtained for estrogenicity at the three different sampling sites for each of the assays in the battery were comparable. In this study we developed, validated and also implemented a battery of assays encompassing both in vitro and in vivo assays, based on different biological mechanisms, to detect estrogenic EDCs. To our knowledge, this is the first study that has used a battery of bioassays to specifically assess a South Africa river for estrogenicity ...

Endocrine disrupting chemicals (EDCs) include a wide variety of structurally diverse compounds that affect normal signaling pathways in most all animal species by multiple mechanisms. Given the global distribution of EDCs in environmental, biological and food samples and commercial products, detection, relative quantitation, and assessment of their biological/toxicological effects is of paramount importance. While instrumental methods allow detection and quantitation of specific (known) EDCs in sample extracts, these methods do not allow identification and characterization of new EDCs. Accordingly, mechanism-based bioanalytical methods have been developed to assist in the identification and characterization of matrices and extracts containing EDCs. The so-called chemically-activated luciferase expression (CALUX) bioassays and related CALUX-type bioassays represent a collection of nuclear receptor-based recombinant cell bioassays for detection of EDCs, including estrogenic-, androgenic- and dioxin-like chemicals. Although CALUX assays are much cheaper and quicker than instrumental analysis and whole animal assays, there is a critical need to make these assays as biologically/toxicologically relevant and consistent with our current understanding of endocrine disruption in order to provide optimal data for selection of chemicals (or mixtures) which warrant in vivo assessment. This includes improved assay characteristics (i.e., minimal detection limits (MDLs)) and response to EDCs, inclusion of more receptors (i.e. multiple subtypes of a given nuclear receptor) and consideration of receptor cross-talk and related mechanisms. In this dissertation, I describe two significantly improved CALUX bioassay systems for EDCs. The first is a set of species-specific Ah receptor (AhR)-responsive CALUX cell lines that exhibit dramatically increased sensitivity (10-100-fold lower MDL (to sub pM concentrations)) and responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related dioxin-like compounds (DLCs), compared to current dioxin-responsive CALUX cell lines. I demonstrate the utility of these novel AhR-responsive CALUX bioassays in analysis of sediment extracts. The second CALUX cell line (estrogen receptor (ER)-CALUX) is a modification of our existing estrogen-responsive human cell line (BG1Luc4E2). Although this cell line has received official regulatory approval from the USEPA and OECD (to the point of being included in the USEPA Endocrine Disruptor Screening Program) and is currently being utilized by the Tox21 program for chemical screening purposes, this cell line only expresses one of the two known human ER subtypes (ER[alpha]). Since both ER subtypes (ER[alpha] and ER[beta]) can differ in their ligand-specificity, cross-talk with other signaling and transcription factors and gene induction profiles, optimal cell bioassay assays should be able to detect the full spectrum of ER-active chemicals (i.e., activators/repressors of ER[alpha] and/or ER[beta]). Accordingly, we stably transfected the ER[alpha]-containing BG1LucE2 CALUX cell line with a human ER[beta] expression vector and screening of the resulting BG1LucER[beta] clones with the ER[beta]-selective ligands genistein and Br-ER[beta]-041 allowed identification and isolation of a clonal cell line (BG1LucER[beta]c9) expressing ER[beta] and highly responsive to ER[beta]-selective ligands. Comparison of the response of BG1Luc4E2 and BG1LucER[beta]c9 cell lines to a chemical library of 176 compounds revealed that the ER[alpha]/ER[beta]-containing BG1LucER[beta]c9 cell line can identify a greater number of potential estrogenic chemicals than the ER[alpha]-containing BG1Luc4E2 cell line. Since cross-talk between the AhR and ER has been identified as a potential mechanism for endocrine disruption in animals exposed to DLCs (which act to repress ER gene signaling), in the final chapter I explored cross-talk between AhR and ER in a human breast carcinoma cell line (SKBR3). I specifically focused on the effect of ER[alpha]/ER[beta] on AhR gene signaling in transiently transfected cells. In SKBR3 cells (which lack functional ER), we observed an ER[alpha] subtype-specific enhancement of AhR-dependent reporter gene expression (from the transfected AhR-responsive plasmid pGudLuc6.1) in the absence of added E2 and a repression of the ER-dependent enhancement of AhR signaling by stimulating transfected ER[alpha] with E2, PPT, or fulvestrant (no repression was observed with ER[beta]). The repressive effect of E2 could be abolished by co-incubation of E2-activated ER[alpha]-transfected SKBR3 cells with the ER antagonist 4-hydroxytamoxifen, demonstrating the requirement for functionally active ER[alpha]. Our ER-dependent repressive results in SKBR3 cells indicate that AhR has selective interactions with ER[alpha] versus ER[beta]. The results presented in this dissertation demonstrate that the CALUX reporter vectors and stable cell lines not only have application for highly sensitive high throughput screening for EDCs systems, but for in-depth mechanistic studies examining crosstalk between receptor signaling pathways which can play a critical role in endocrine disruption.

Endocrine disrupting chemicals (EDCs) are ubiquitous in the environment and their presence in water bodies is documented. They discharge into surface water (SW) unmonitored, posing a threat to both aquatic and terrestrial lives. This is a challenge as not all populations have access to treated drinking water (TDW). The EDC contaminated serves as a route of exposure, together with ineffective treatment plants. Given the complexity of the endocrine system, EDCs may mimic or antagonise natural hormones or disrupt their synthesis, metabolism and excretion. The associated health effects include testicular dysgenesis syndrome, metabolic disorders and cancers. Policy and internationally standardised test methods are however sti ll limited. This study therefore aimed to assess the suitability of two assays used for screening estrogenic activity and one for androgenic activity in different water sources. The study consisted of two phases. In phase 1, water sample (tap, surface and treated wastewater) were collected from a catchment area in Pretoria. The samples and a spiked MilliQ laboratory water sample were extracted with solid phase extraction (SPE) and sent to Germany for distribution to participating laboratories. Samples (n=24) from six different countries were received to test for androgenic activity in the MDA-kb2 reporter gene assay. In phase 2, SW and TDW samples were collected from April 2015 until March 2016. The samples were filtered, extracted using SPE and assayed with the YES assay, T47D-KBluc reporter gene assay for estrogenic activity and MDA-kb2 reporter gene assay for androgenic activity. In phase 1, androgenic activity was detected in 4 out of 24 (21%) samples and ranged from 0.23 ± 0.040 ng/L to 0.008 ± 0.001 ng/L DHTEqs. In phase 2, estrogenic activity was detected in 16 out of 24 (67%) SW samples in the T47DKBluc reporter gene assay and ranged from 0.31 ± 0.05 pg/L to 10.51 ± 5.74 pg/L EEqs. It was below the detection limit (dl) in the YES assay. Androgenic activity was detected in 4 out of 24 (17%) SW samples, ranging from 0.0033 ± 0.0050 ng/L to 0.090 ± 0.040 ng/L DHTEqs. Androgenic and estrogenic activity was higher i n pretreatment samples compared to post-treatment in both treatment plants. In phase 1, the MDA-kb2 reporter gene assay was successfully applied to water samples from different sources. Androgenic activity was highest in treated wastewater. In phase 2, treatment plants proved to be effective in removing estrogens detected in the SW samples, as the TDW samples were below the dl. Estrogenic activity is within the ranges reported in other studies. Positive samples were below the 0.7 ng/L proposed trigger value for health risk assessments. Detected androgenic activity was lower in TDW samples compared to the SW samples supplying the two treatment plants indicating that they were both effective in removing the androgenic activity detected. Few studies have reported androgenic activity in tap water. This study strengthens the argument for using a battery of assays when monitoring endocrine activity as EDCs occur at low concentrations in mixtures.

Enables researchers to assess the effects of endocrine disrupters as well as comply with new environmental regulations Endocrine disrupters are chemicals both man-made and natural that interfere with the body's endocrine system, potentially resulting in adverse developmental, reproductive, neurological, and immune effects. In recent years, a number of regulatory authorities around the world have drafted or enacted legislation that requires the detection and assessment of the effects of endocrine disrupters on both humans and wildlife. In response, this book provides comprehensive, up-to-date information on the latest tested and proven methods used to detect and assess the environmental hazards posed by endocrine-disrupting chemicals. Endocrine Disrupters is divided into chapters covering each major taxon as well as chapters dedicated to hazard assessment and regulation. The book covers testing methods for all the vertebrate groups and several invertebrate phyla, including: Crustaceans and mollusks Insects Fish Amphibians and reptiles Birds and mammals Moreover, the book emphasizes practical, ethical testing methods that combine sensitivity, efficiency, statistical power, and reasonable cost. Each chapter is written by one or more international experts in ecotoxicology, offering readers step-by-step guidance for implementing each method based on the latest research and the authors' firsthand laboratory experience. Furthermore, all the chapters have been subjected to a rigorous peer review and edited in light of the reviewers' comments. References at the end of each chapter guide readers to the literature in the field. Endocrine Disrupters is recommended for scientists who need to test chemicals for possible endocrine-disrupting properties. It is also recommended for regulatory authorities who need to decide whether particular chemicals can be safely marketed.

To assure safe levels of human exposure to chemicals, in toxicological risk assessment advances are made to shift from using animal-based to animal-free testing strategies. This is fuelled by ethical, economic, and legislative issues and the scientific rationale that experimental animals do not adequately represent the human body and animal test guidelines may not cover human pathologies. In Next Generation Risk Assessment (NGRA), the development and use of non-animal based new approach methodologies (NAMs) aims not to predict chemical-induced pathologies in animals but to assure human safety, eliminating the need for animal data. The aim of this thesis to perform NGRA to inform human-relevant safe levels of chemical exposure, integrating in vitro-in silico approaches for chemicals with putative (anti)androgenic and/or estrogenic effects. NGRA can use the Dietary Comparator Ratio (DCR) to evaluate the safety of a defined exposure to a compound with an identified mode of action. The DCR compares the Exposure Activity Ratio (EAR) for the compound of interest, to the EAR of an established safe level of human exposure to a comparator compound with the same mode of action. A DCR ≤ 1 indicates the exposure evaluated is safe. It was aimed to define adequate and safe comparator compound exposures for evaluation of endocrine effects. 3,3-Diindolylmethane (DIM), from cruciferous vegetables, and the anti-androgenic drug bicalutamide (BIC) were used to evaluate anti-androgenic effects and the EAR values for these comparator compounds were defined using an in vitro androgenic reporter gene assay. The isoflavone genistein (GEN) was used to evaluate estrogenic effects and its EAR value was set using in vitro estrogenic proliferation and reporter gene assays. The EAR values of DIM and BIC were evaluated using physiologically based kinetic (PBK) modelling and comparison to in vivo reported safe and therapeutic-active levels, respectively. The EAR values of GEN were evaluated by comparison with in vivo reported safe internal levels of GEN following different diets. The adequacy of the comparator EAR values was further evaluated by comparing the generated DCRs of a series of test compound exposures to actual knowledge of their safety regarding in vivo anti-androgenicity or estrogenicity. Exposure scenarios of nine anti-androgenic pesticides, phenols, and drugs and of 12 estrogenic hormones, phytoestrogens, pesticides, phenols, parabens, phthalates, and drugs were included. Results obtained supported the use of the in vitro bioactivity assay-derived comparator EARs for DCR-based NGRA for putative anti-androgenic and estrogenic compounds. This further validates the DCR approach as an important animal free tool in NGRA and will further support the safety assessment of endocrine acting substances. Another NGRA strategy was explored aiming at including hepatic biotransformation by providing a proof of principle for PBK modelling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) using a toxic equivalency factor (TEF) approach. In the PBK modelling-facilitated QIVIVE of the selected anti-androgenic model compound flutamide (FLU), its in vitro AR-CALUX derived concentration-response curve was translated to the corresponding in vivo dose-response curves, either excluding or including the activity of its more anti-androgenic active metabolite hydroxyflutamide (HF). It was demonstrated that including the bioactivity of the active metabolite in PBK modelling-facilitated QIVIVE provides a better and more appropriate point of departure (PoD) to assure human safe exposure levels to the parent compound, whereas excluding the activity of the metabolite will potentially result in an underestimation of the corresponding risk. Finally, a two-chamber co-culture system with human liver and reporter cells was developed to assess androgenic responses in the absence and presence of a biotransformation system. The androgenic response of testosterone (T) and 5α-dihydrotestosterone (DHT), which are hepatically detoxified, were assessed using the AR-CALUX or AR-INDIGO assay in the absence or presence of 3D HepaRG microtissues, that exhibit active hepatic metabolism. Androstenedione, which is an inactivated metabolite, was formed following incubation of T with the 3D HepaRG microtissues which translated in the co-culture system to a significantly reduced T mediated induction of the AR response in both reporter gene assays in the presence of 3D HepaRG microtissues. A similar 3D HepaRG microtissues dependent reduction in the DHT induced AR response was observed in line with the hepatic inactivation of DHT by a similar biotransformation step. The two-chamber co-culture system with human liver and reporter cells may thus provide a screening tool to identify chemicals for which hepatic metabolism affects their bioactivity and thus should be included when setting the PoD of the parent compound. In conclusion, the DCR approach provides an appropriate NAM for the safety assessment of human chemical exposures to anti-androgenic and estrogenic compounds. Furthermore, biologically active metabolites should be taken into account in the PBK modelling-facilitated QIVIVE for evaluating the safety of a parent compound. Lastly, the two-chamber co-culture system with human liver and reporter cells is a new technology that will allow the identification of hepatic metabolites affecting the in vitro bioactivity of a parent compound. Taken together, it is concluded that the NGRA strategies presented in this thesis are appropriate and of value for human safety assessment of exposure to (anti)androgens and estrogens in an animal free in silico/in vitro 3R compliant way.