Factors affecting the removal of organic micropollutants from wastewater in conventional treatment plants (CTP) and membrane bioreactors (MBR)

As a consequence of insufficient removal during treatment of wastewater released from industry and households, different classes of organic micropollutants are nowadays detected in surface and drinking water. Among these micropollutants, bioactive substances, e.g., endocrine disrupting compounds and pharmaceuticals, have been incriminated in negative effects on living organisms in aquatic biotope. Much research was done in the last years on the fate and removal of those compounds from wastewater. An important point it is to understand the role of applied treatment conditions (sludge retention time (SRT), biomass concentration, temperature, pH value, dominant class of micropollutants, etc.) for the efficiency of conventional treatment plants (CTP) and membrane bioreactors (MBR) concerning the removal of micropollutants such as pharmaceuticals, steroid- and xeno-estrogens. Nevertheless, the removal rates differ even from one compound to the other and are related to the physico-chemical characteristics of the xenobiotics.     

Analysis of the stress effects of endocrine disrupting chemicals (EDcs) on Escherichia coli

In this study, three of the representative EDCs, 17beta-estradiol, bisphenol A, and styrene, were employed to find their mode of toxic actions in E. coli. To accomplish this, four different stress response genes, recA, katG, fabA, and grpE genes, were used as a representative for DNA, oxidative, membrane, or protein damage, respectively. The expression levels of these four genes were quantified using a real-time RT-PCR after challenge with three different EDCs individually. Bisphenol A and styrene caused high-level expression of recA and katG genes, respectively, whereas 17beta-estradiol made no significant changes in expression of any of those genes. These results lead to the classification of the mode of toxic actions of EDCs on E. coli.     

Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals

Laccase from the white rot fungus Coriolopsis polyzona was immobilized for the first time through the formation of cross-linked enzyme aggregates (CLEAs). Laccase CLEAs were produced by using 1000g of polyethylene glycol per liter of enzyme solution as precipitant and 200muM of glutaraldehyde as a cross-linking agent. These CLEAs had a laccase activity of 148Ug(-1) and an activity recovery of 60.2% when using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate. CLEAs formed by co-aggregation with bovine serum albumin (BSA) as a stabilizer showed lower laccase activity and affinity for ABTS than those without BSA. The CLEAs co-aggregated with BSA showed higher residual activity against a protease, NaN(3), EDTA, methanol and acetone. The thermoresistance was higher for CLEAs than for free laccase and also higher for CLEAs co-aggregated with BSA than for simple CLEAs when tested at a pH of 3 and a temperature of 40 degrees C. Finally, laccase CLEAs were tested for their capacity to eliminate the known or suspected endocrine disrupting chemicals (EDCs) nonylphenol, bisphenol A and triclosan in a fluidized bed reactor. A 100-ml reactor with 0.5mg of laccase CLEAs operated continuously at a hydraulic retention time of 150min at room temperature and pH 5 could remove all three EDCs from a 5mgl(-1) solution.     

Toxicity monitoring of endocrine disrupting chemicals (EDCs) using freeze-dried recombinant bioluminescent bacteria

Five different freeze-dried recombinant bioluminescent bacteria were used for the detection of cellular stresses caused by endocrine disrupting chemicals. These strains were DPD2794 (recA::luxCDABE), which is sensitive to DNA damage, DPD2540 (fabA::luxCDABE), sensitive to cellular membrane damage, DPD2511 (katG::luxCDABE), sensitive to oxidative damage, and TV1061 (grpE::luxCDABE), sensitive to protein damage. GC2, which emits bioluminescence constitutively, was also used in this study. The toxicity of several chemicals was determined on the first four freeze-dried bacteria, while nonspecific cellular stresses were measured using GC2. Damage caused by known endocrine disrupting chemicals, such as nonyl phenol, bisphenol A, and styrene, was detected and classified according to toxicity mode, while others, such as phathalate and DDT, were not detected with the bacteria. These results suggest that endocrine disrupting chemicals are toxic in bacteria, and do not act via an estrogenic effect, and that toxicity monitoring and classification of some endocrine disrupting chemicals may be possible in the field using these freeze-dried recombinant bioluminescent bacteria.     

Non-traditional targets of endocrine disrupting chemicals: the roots of hormone signaling

The topic of endocrine disruption and the broad range of physiologicaleffects caused by endocrine disrupting chemicals (EDCs) canonly be meaningfully framed within an ecological and evolutionarycontext. Environmental pollutants and EDCs operate by disruptingthe "chemical communication" that coordinates signaling withinan organism. Here we discuss how EDCs are also able to disruptthe chemical communication between plants and soil bacterianecessary for initiating nitrogen-fixing symbiosis. We alsoexamine, through examples of pollutant-related impacts on awide range of invertebrates, the need for identifying emergingtargets of EDCs. We suggest broadening the defined field ofendocrine disruption to encompass the effects of synthetic chemicalsthat interfere with signaling and communication, not only withinan organism, but also between organisms and linking ecosystems.The ecological consequences of failing to recognize novel targetsof chemical pollutants and EDCs may be a net loss of biologicaldiversity and a further imbalance of the global nitrogen cycle.     

Neuroendocrine and behavioral implications of endocrine disrupting chemicals in quail

Studies in our laboratory have focused on endocrine, neuroendocrine, and behavioral components of reproduction in the Japanese quail. These studies considered various stages in the life cycle, including embryonic development, sexual maturation, adult reproductive function, and aging. A major focus of our research has been the role of neuroendocrine systems that appear to synchronize both endocrine and behavioral responses. These studies provide the basis for our more recent research on the impact of endocrine disrupting chemicals (EDCs) on reproductive function in the Japanese quail. These endocrine active chemicals include pesticides, herbicides, industrial products, and plant phytoestrogens. Many of these chemicals appear to mimic vertebrate steroids, often by interacting with steroid receptors. However, most EDCs have relatively weak biological activity compared to native steroid hormones. Therefore, it becomes important to understand the mode and mechanism of action of classes of these chemicals and sensitive stages in the life history of various species. Precocial birds, such as the Japanese quail, are likely to be sensitive to EDC effects during embryonic development, because sexual differentiation occurs during this period. Accordingly, adult quail may be less impacted by EDC exposure. Because there are a great many data available on normal development and reproductive function in this species, the Japanese quail provides an excellent model for examining the effects of EDCs. Thus, we have begun studies using a Japanese quail model system to study the effects of EDCs on reproductive endocrine and behavioral responses. In this review, we have two goals: first, to provide a summary of reproductive development and sexual differentiation in intact Japanese quail embryos, including ontogenetic patterns in steroid hormones in the embryonic and maturing quail. Second, we discuss some recent data from experiments in our laboratory in which EDCs have been tested in Japanese quail. The Japanese quail provides an excellent avian model for testing EDCs because this species has well-characterized reproductive endocrine and behavioral responses. Considerable research has been conducted in quail in which the effects of embryonic steroid exposure have been studied relative to reproductive behavior. Moreover, developmental processes have been studied extensively and include investigations of the reproductive axis, thyroid system, and stress and immune responses. We have conducted a number of studies, which have considered long-term neuroendocrine consequences as well as behavioral responses to steroids. Some of these studies have specifically tested the effects of embryonic steroid exposure on later reproductive function in a multigenerational context. A multigenerational exposure provides a basis for understanding potential exposure scenarios in the field. In addition, potential routes of exposure to EDCs for avian species are being considered, as well as differential effects due to stage of the life cycle at exposure to an EDC. The studies in our laboratory have used both diet and egg injection as modes of exposure for Japanese quail. In this way, birds were exposed to a specific dose of an EDC at a selected stage in development by injection. Alternatively, dietary exposure appears to be a primary route of exposure; therefore experimental exposure through the diet mimics potential field situations. Thus, experiments should consider a number of aspects of exposure when attempting to replicate field exposures to EDCs.     

Membrane bioreactors for the removal of anionic micropollutants from drinking water

Biological treatment processes allow for the effective elimination of anionic micropollutants from drinking water. However, special technologies have to be implemented to eliminate the target pollutants without changing water quality, either by adding new pollutants or removing essential water components. Some innovative technologies that combine the use of membranes with the biological degradation of ionic micropollutants in order to minimize the secondary contamination of treated water include pressure-driven membrane bioreactors, gas-transfer membrane bioreactors and ion exchange membrane bioreactors.     

Immobilization of laccase from the white rot fungus Coriolopsis polyzona and use of the immobilized biocatalyst for the continuous elimination of endocrine disrupting chemicals

Laccase from the white rot fungus strain Coriolopsis polyzona was immobilized covalently on the diatomaceous earth support Celite^® R-633 using different strategies. A first methodology involved the sequential activation of the support surface with γ-aminopropyltriethoxysilane followed by the reaction of the functionalized surface with glutaraldehyde (GLU) or glyoxal (GLY) and the immobilization of laccase on the activated surface. Another strategy tested the simultaneous internal cross-linking of the protein with GLU or GLY and the immobilization of the laccase on the silanized surface. Finally, these two strategies were modified to test the impact of the concomitant addition of bovine serum albumin (BSA) as a stabilizing agent during the immobilization steps. The highest laccase activity and the greatest degree of activity recovery (tested using 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as the substrate) were achieved by the sequential immobilization procedure using GLU as the cross-linking agent. The solid catalysts featuring internal cross-linking of the protein showed significantly higher stability against several denaturants. The Michaelis–Menten kinetic parameters with respect to ABTS revealed a higher affinity for this substrate in the case of the sequential procedure compared to the simultaneous approach. The biocatalyst formed using GLU in the sequential procedure was applied in a packed bed reactor for the continuous treatment of 5 mg l⁻¹ solutions of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA) and triclosan (TCS) through repeated batch treatments. All of these EDCs could be eliminated at a contact time of less than 200 min by using, respectively, 3.75 units (U) of laccase activity for BPA and TCS and 1.88 U for NP. These performances of elimination were maintained over five consecutive treatment cycles using the same biocatalyst. This system could also remove these EDCs from 100 mg l⁻¹ solutions. The Michaelis–Menten kinetic parameters with respect to these chemicals showed a decreasing affinity of the solid biocatalyst for NP, TCS and BPA in that order.     

Comparison of two fluorescence immunoassay methods for the detection of endocrine disrupting chemicals in water

We describe two fluorescence immunoassays capable of detecting endocrine disrupting compounds in waste water. The first fluorescence method is a heterogeneous assay using total internal reflection fluorescence (TIRF) detection. The second method is a homogeneous assay that utilizes energy transfer (ETIA). Both fluorescence immunoassays are compared with respect to detection principle and ability to quantify the model analytes estrone, estradiol, and ethinylestradiol in a complex matrix regarding recovery rates and limits of detection. Calibrations were performed for the three analytes using both fluorescence methods. Limits of detection between 0.01 and 0.85 microg/l are achieved. In addition, measurements in synthetic waste water spiked with the analytes were performed. Both immunoassays allow the detection in waste water with recovery rates in the range of 70-112%.     

Enhanced expression of laccase during the degradation of endocrine disrupting chemicals in Trametes versicolor

A putative laccase cDNA from a white-rot basidiomycete, Trametes versicolor, that consisted of 1,769 nucleotides was cloned using the rapid amplification of cDNA ends (RACE)-PCR method. The deduced amino acid sequence had 4 putative copper binding regions, which are common to fungal laccases. In addition, the sequence was 57 approximately 97 % homologous to sequences of other T. versicolor laccases. Additionally, the expression of laccase and manganese peroxidase in this fungus were both greatly increased under degrading conditions for bisphenol A, nonylphenol and two phthalic esters (benzylbutylphthalate and diethylphthalate), all of which are reportedly endocrine disrupting chemicals (EDCs). Furthermore, the estrogenic activities of the EDCs also decreased rapidly during incubation when examined in a two-hybrid yeast system. Finally, kojic acid inhibited the removal of estrogenic activities generated by bisphenol A and nonylphenol, which confirmed that laccase was involved in the degradation of EDCs in T. versicolor.     

Application of the OECD 301F respirometric test for the biodegradability assessment of various potential endocrine disrupting chemicals

The biodegradability of several potential endocrine disrupting compounds, namely 4-n-nonylphenol (4-n-NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), bisphenol A (BPA), triclosan (TCS), di-(2-ethylhexyl)-phthalate (DEHP), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) was evaluated in this study, using OECD method 301F (manometric respirometry test) and activated sludge as inoculum. According to the results, 4-n-NP and BPA meet the strict definition of ready biodegradability and they are not expected to be persistent during the activated sludge process. Partial biodegradation was observed for DEHP (58.7+/-5.7%, n=3), TCS (52.1+/-8.5%, n=3) and NP1EO (25.9+/-8.1%, n=3), indicating their possible biodegradation in wastewater treatment systems, while no biodegradation was observed for NP2EO, PFOA and PFNA. Experiments in the co-presence of a readily biodegradable compound showed the absence of co-metabolic phenomena during 4-n-NP, BPA and TCS biodegradation. Using first order kinetics to describe biodegradation of the target compounds, half-lives of 4.3+/-0.6, 1.3+/-0.2, 1.8+/-0.5, 6.9+/-2.6 days were calculated for 4-n-NP, BPA, TCS and DEHP, respectively. Toxicity tests using marine bacterium Vibrio fischeri showed that biodegradation of 4-n-NP, NP1EO, BPA and TCS is a simultaneous detoxification process, while possible abiotic or biotic transformations of NP2EO, DEHP, PFOA and PFNA during respirometric test resulted to significant increase of their toxicities.     

Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor

Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, k_cat/K_M) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was attained by inactivated laccase.     

p53 gene expression is modulated by endocrine disrupting chemicals in the hermaphroditic fish, Kryptolebias marmoratus

The full-length of cDNA of tumour suppressor gene p53 from the self-fertilizing fish Kryptolebias marmoratus (Km-p53) was determined using molecular cloning and rapid amplification of cDNA ends (RACE). The Complete cDNA sequences of K. marmoratus (Km-p53) gene was 1.8 kb in length. K. marmoratus p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. Although basal level of expression of Km-p53 mRNA was low, all the studied tissues showed some level of expression. After exposure of K. marmoratus to endocrine disrupting chemicals (EDCs) such as bisphenol A, 4-nonylphenol, and 4-tert-octylphenol, Km-p53 expression was significantly increased within 3 h of exposure in juveniles. However, expression was down-regulated by exposure to most of the EDCs when measured at 96 h in adult fish. In adult fish, suppressive effect of EDCs was more pronounced in liver as compared to other tissues. These findings suggest that Km-p53 gene would be involved in cellular defense mechanism in early stage of exposure to EDCs and long-term exposure may suppress its expression. It may be possible that the suppression of p53 by EDCs may predispose the host to environmental chemical carcinogenesis.     

Development of comparative methods using gas chromatography-mass spectrometry and capillary electrophoresis for determination of endocrine disrupting chemicals in bio-solids

Two analytical separation techniques are being investigated for their potential in determining a wide range of endocrine disrupting chemicals (EDCs) in the environment. Capillary electrophoresis (CE) in the micellar mode in conjunction with a cyclodextrin (CD) modifier is shown to have potential for determination of alkylphenol breakdown products. Gas chromatography with mass spectrometric (GC-MS) detection is being utilised for validation of the CE method development and in addition as a separation technique to optimise preconcentration using solid-phase extraction. GC has demonstrated potential for the separation of 26 priority chemicals suspected as being endocrine disrupting compounds. The challenge of the method development process lies in the fact that these compounds are of differing polarities, size and charge and therefore are difficult to separate in a single run. Capillary electrophoresis in the CD-MEKC (micellar electrokinetic chromatography) mode is showing potential in this regard. Limits of determination are in the low mg/l range for CE and GC, however, using preconcentration it is possible to improve detection sensitivity with >80% recovery for some analytes and up to 100% recovery for most target species.     

Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.