LANCE: Laccase-nanoparticle conjugates for the elimination of micropollutants (endocrine disrupting chemicals) from wastewater in bioreactors

Elimination of recalcitrant chemicals during wastewater treatment is a difficult problem for both developing and industrialized countries. The biological elimination of very persistent xenobiotics such as endocrine disrupting chemicals from municipal and industrial sewage treatment plants is an ambitious challenge as existing physico-chemical methods, such as advanced oxidation processes, are energy-intensive and consume high amounts of chemicals. Through the entry into force of strict legislative measures, such as the Water Framework Directives (EU WFD in Directive 2000/60/EC of the European Parliament and of the Council establishing a framework for the Community action in the field of water policy, 2000) and REACH (REACH EU in European Community Regulation on chemicals and their safe use (EC 1907/2006), 2007), the market for wastewater treatment is exploding. For instance the European market potential for the membrane bioreactor technology is estimated to 57 M€ per year. Based on recent progresses in nanotechnology, new developments in catalysis and environmental applications can be foreseen for the near future. Indeed, because of high surface area-to-volume ratio in nano-systems, heterogeneous enzymatic or catalytic reactions can be greatly enhanced. In the LANCE project a nanoparticle (NP)-based technology is under development. Cheap and resistant oxidative enzymes, i.e. laccases are immobilized onto the surface of the particles in order to produce systems possessing a broad substrate spectrum for the degradation of cocktails of recalcitrant pollutants. One of the objectives is to produce NPs that are compatible with wastewater treatment and can be synthesised in a cost-effective and large-scale fashion, e.g. silica-based NPs using flame spray pyrolysis and emulsion-based techniques. The modified particles are applied in bioreactors where they are retained, i.e. membrane bioreactors or perfusion basket reactors to eliminate pollutants from the wastewater. Such reactors allow multi-cycle use of the NPs coated with active enzymes and thus contribute to decrease the treatment costs. The two-year activities of the LANCE project encompass the synthesis of various NP systems, the immobilization of selected low cost industrial laccases on the latter, and the technical and scientific proof of the “depollution” concept.     

Facile and rapid magnetic relaxation switch immunosensor for endocrine-disrupting chemicals

In order to develop facile, fast and sensitive detection methods for endocrine-disrupting chemicals (EDCs), we described a sensitive biosensing system involving magnetic relaxation switch, based on the assembly of cross-linked superparamagnetic iron oxide (CLIO) nanoparticles induced by the antigen-antibody biorecognition. The design of smart CLIO-based superparamagnetic iron oxide nanoparticles and antigen-OVA was described for the detection of bisphenol A [2,2-bis (4-hydroxyphenol) propane (BPA)]. The addition of BPA to the rapid magnetic relaxation switch immunosensor led to transverse relaxation time (T2) shortening compared to a blank control as shown by NMR relaxometry measurements. This process was also applied to the rapid and facile determination of concentrations of BPA in drinking water (tap water). Good linearity for all calibration curves was obtained, and the limit of detection (LOD) for BPA was 0.4 ng/mL in tap water.     

Sediments in the mangrove areas contribute to the removal of endocrine disrupting chemicals in coastal sediments of Macau SAR,China, and harbour microbial communities capable of degrading E2, EE2, BPA and BPS

The occurrence of endocrine disrupting chemicals (EDCs) is a major issue for marine and coastal environments in the proximity of urban areas. The occurrence of EDCs in the Pearl River Delta region is well documented but specific data related to Macao is unavailable. The levels of bisphenol-A (BPA), estrone (E1), 17α-estradiol (αE2), 17β-estradiol (E2), estriol (E3), and 17α-ethynylestradiol (EE2) were measured in sediment samples collected along the coastline of Macao. BPA was found in all 45 collected samples with lower BPA concentrations associated to the presence of mangrove trees. Biodegradation assays were performed to evaluate the capacity of the microbial communities of the surveyed ecosystems to degrade BPA and its analogue BPS. Using sediments collected at a WWTP discharge point as inoculum, at a concentration of 2 mg l^?1 complete removal of BPA was observed within 6 days, whereas for the same concentration BPS removal was of 95% after 10 days, which is particularly interesting since this compound is considered recalcitrant to biodegradation and likely to accumulate in the environment. Supplementation with BPA improved the degradation of bisphenol-S (BPS). Aiming at the isolation of EDCs-degrading bacteria, enrichments were established with sediments supplied with BPA, BPS, E2 and EE2, which led to the isolation of a bacterial strain, identified as Rhodoccoccus sp. ED55, able to degrade the four compounds at different extents. The isolated strain represents a valuable candidate for bioremediation of contaminated soils and waters.

     

Assessment of human contamination of estrogenic endocrine-disrupting chemicals and their risk for human reproduction

There is broad human exposure to estrogenic endocrine-disrupting chemicals (EDCs), but the data sets that exist are primarily for various environmental media such as food and water rather than the most relevant internal exposure. We have detected various kind of EDC contamination in humans including dioxin and bisphenol A (BPA) widely used for the production of plastic products. BPA was present in serum and follicular fluid at approximately 1–2 ng/ml, as well as in fetal serum and full-term amniotic fluid, confirming passage through the placenta. An approximately five-fold higher concentration, 8.3 ± 8.7 ng/ml, was revealed in amniotic fluid at 15–18 weeks of gestation, compared to other fluids showing increased exposure at the critical developmental period in humans. Interestingly, serum BPA concentrations were significantly higher in normal men and in women with polycystic ovary syndrome (PCOS) compared with normal women possibly due to differences in the androgen-related metabolism of BPA. These findings may provide some insight into the metabolism of EDCs in human and the pathophysiology of endocrine disorders such as PCOS. Dioxin contamination in relationship to development of endometriosis is also discussed.     

Potential applications of the oxidoreductive enzymes in the decolorization and detoxification of textile and other synthetic dyes from polluted water: a review

Recently, the enzymatic approach has attracted much interest in the decolorization/degradation of textile and other industrially important dyes from wastewater as an alternative strategy to conventional chemical, physical and biological treatments, which pose serious limitations. Enzymatic treatment is very useful due to the action of enzymes on pollutants even when they are present in very dilute solutions and recalcitrant to the action of various microbes participating in the degradation of dyes. The potential of the enzymes (peroxidases, manganese peroxidases, lignin peroxidases, laccases, microperoxidase-11, polyphenol oxidases, and azoreductases) has been exploited in the decolorization and degradation of dyes. Some of the recalcitrant dyes were not degraded/decolorized in the presence of such enzymes. The addition of certain redox mediators enhanced the range of substrates and efficiency of degradation of the recalcitrant compounds. Several redox mediators have been reported in the literature, but very few of them are frequently used (e.g., 1-hydroxybenzotriazole, veratryl alcohol, violuric acid, 2-methoxy-phenothiazone). Soluble enzymes cannot be exploited at the large scale due to limitations such as stability and reusability. Therefore, the use of immobilized enzymes has significant advantages over soluble enzymes. In the near future, technology based on the enzymatic treatment of dyes present in the industrial effluents/wastewater will play a vital role. Treatment of wastewater on a large scale will also be possible by using reactors containing immobilized enzymes.     

Potential Applications of the Oxidoreductive Enzymes in the Decolorization and Detoxification of Textile and Other Synthetic Dyes from Polluted Water: A Review

ABSTRACT

Recently, the enzymatic approach has attracted much interest in the decolorization/degradation of textile and other industrially important dyes from wastewater as an alternative strategy to conventional chemical, physical and biological treatments, which pose serious limitations. Enzymatic treatment is very useful due to the action of enzymes on pollutants even when they are present in very dilute solutions and recalcitrant to the action of various microbes participating in the degradation of dyes. The potential of the enzymes (peroxidases, manganese peroxidases, lignin peroxidases, laccases, microperoxidase-11, polyphenol oxidases, and azoreductases) has been exploited in the decolorization and degradation of dyes. Some of the recalcitrant dyes were not degraded/decolorized in the presence of such enzymes. The addition of certain redox mediators enhanced the range of substrates and efficiency of degradation of the recalcitrant compounds. Several redox mediators have been reported in the literature, but very few of them are frequently used (e.g., 1-hydroxybenzotriazole, veratryl alcohol, violuric acid, 2-methoxy-phenothiazone). Soluble enzymes cannot be exploited at the large scale due to limitations such as stability and reusability. Therefore, the use of immobilized enzymes has significant advantages over soluble enzymes. In the near future, technology based on the enzymatic treatment of dyes present in the industrial effluents/wastewater will play a vital role. Treatment of wastewater on a large scale will also be possible by using reactors containing immobilized enzymes.     

The naringenin-induced proapoptotic effect in breast cancer cell lines holds out against a high bisphenol a background

Fruit and vegetable consumption has generally been associated with the prevention or suppression of cancer. However, food could contain a multitude of chemicals (e.g., bisphenol A; BPA) that could synergize or antagonize the effects of diet-derived compounds. Remarkably, food containers (e.g., water and infant bottles) are the largest source of exposure to BPA for human beings. Here, the effects of the coexposure of naringenin (Nar, 1.0 × 10(-9) M to 1.0 × 10(-4) M) and BPA (1.0 × 10(-5) M) in estrogen-dependent breast cancer cell lines expressing (i.e., MCF-7 and T47D) or not expressing (i.e., MDA-MB-231) estrogen receptor α (ERα) are reported. Although both Nar and BPA bind to ERα, they induce opposite effects on breast cancer cell growth. BPA induces cell proliferation, whereas Nar only decreases the number of ERα-positive cells (i.e., MCF-7 and T47D). Notably, even in the presence of BPA, Nar impairs breast cancer cell proliferation by activating caspase-3. The molecular pathways involved require p38 activation, whereas, the BPA-induced AKT activation is completely prevented by the Nar treatment. As a whole, Nar maintains its proapoptotic effects even in the presence of the food contaminant BPA, thus, enlarging the chemopreventive potential of this flavanone.     

Endocrine disrupting chemicals and disease susceptibility

Environmental chemicals have significant impacts on biological systems. Chemical exposures during early stages of development can disrupt normal patterns of development and thus dramatically alter disease susceptibility later in life. Endocrine disrupting chemicals (EDCs) interfere with the body's endocrine system and produce adverse developmental, reproductive, neurological, cardiovascular, metabolic and immune effects in humans. A wide range of substances, both natural and man-made, are thought to cause endocrine disruption, including pharmaceuticals, dioxin and dioxin-like compounds, polychlorinated biphenyls, DDT and other pesticides, and components of plastics such as bisphenol A (BPA) and phthalates. EDCs are found in many everyday products--including plastic bottles, metal food cans, detergents, flame retardants, food additives, toys, cosmetics, and pesticides. EDCs interfere with the synthesis, secretion, transport, activity, or elimination of natural hormones. This interference can block or mimic hormone action, causing a wide range of effects. This review focuses on the mechanisms and modes of action by which EDCs alter hormone signaling. It also includes brief overviews of select disease endpoints associated with endocrine disruption.     

What is an endocrine disruptor?

Endocrine disrupting chemicals (EDCs) and potential EDCs are mostly man-made found in various materials. By interfering with the body's endocrine system, endocrine disruptors produce adverse developmental, reproductive, neurological, and immune effects in humans, abnormal growth patterns and neurodevelopmental delays in children. Thus, diethylstilbestrol (DES) a non-steroidal estrogen, which is regarded as a proof of concept, induces clear cell carcinoma among young women. EDCS may be found in plastic bottles and metal food cans (BPA), medical devices (phthalates), detergents, flame retardants (polybrominated diphenyl ethers), food (BPA), toys (phthalates), cosmetics and drugs (parabens), and pesticides (alkyl phenols such as nonylphenol). The deleterious effects of endocrine disruptors constitute a real public health issue. However concerning the mechanisms of action of EDCs, many questions remain unanswered and need further investigations.     

Biological monitoring of bisphenol A with HLPC/FLD and LC/MS/MS assays

Biological monitoring is a necessary process for risk assessment of endocrine disrupting chemicals (EDCs), particularly, bisphenol A (BPA), in breast milk, because its human risks are not clear yet, and infants, who feed on breast milk, are highly susceptible for EDCs. Concerning biological monitoring of BPA, the HPLC/FLD has been widely used before the LC/MS/MS. However, there was no report, which simultaneously evaluated the two methods in real analyses. Therefore, we analyzed BPA with LC/MS/MS and HPLC/FLD in human breast milk and conducted comparison of two methods in analyzed BPA levels. After establishing optimal condition, e.g. linearity, recovery, reproducibility and free BPA system, we analyzed BPA levels in human breast milk samples (N = 100). The LOQs were similar in the two methods, i.e. 1.8 and 1.3 ng/mL for the HPLC/FLD and LC/MS/MS assays, respectively. There were strong associations between total BPA levels with the two methods (R² = 0.40, p < 0.01), however, only 11% of them were analyzed as similar levels with 15% CVs. In addition, the detection range of BPA was broader in the HPLC method than the LC/MS/MS method. However, the BPA levels in the HPLC/FLD analysis were lower than those in the LC/MS/MS analysis (p < 0.01). Thus, the differences in BPA levels between the two methods may come from mainly over-estimation with the LC/MS/MS method in low BPA samples and some of poor resolution with the HPLC/FLD in high BPA samples.     

Effects of developmental exposure to bisphenol A and ethinyl estradiol on spatial navigational learning and memory in painted turtles (Chrysemys picta)

Developmental exposure of turtles and other reptiles to endocrine disrupting chemicals (EDCs), including bisphenol A (BPA) and ethinyl estradiol (EE2, estrogen present in birth control pills), can induce partial to full gonadal sex-reversal in males. No prior studies have considered whether in ovo exposure to EDCs disrupts normal brain sexual differentiation. Yet, rodent model studies indicate early exposure to these chemicals disturbs sexually selected behavioral traits, including spatial navigational learning and memory. Thus, we sought to determine whether developmental exposure of painted turtles (Chrysemys picta) to BPA and EE2 results in sex-dependent behavioral changes. At developmental stage 17, turtles incubated at 26⁰C (male-inducing temperature) were treated with 1) BPA High (100 μg /mL), 2) BPA Low (0.01 μg/mL), 3) EE2 (0.2 μg/mL), or 4) vehicle or no vehicle control groups. Five months after hatching, turtles were tested with a spatial navigational test that included four food containers, only one of which was baited with food. Each turtle was randomly assigned one container that did not change over the trial period. Each individual was tested for 14 consecutive days. Results show developmental exposure to BPA High and EE2 improved spatial navigational learning and memory, as evidenced by increased number of times spent in the correct target zone and greater likelihood of solving the maze compared to control turtles. This study is the first to show that in addition to overriding temperature sex determination (TSD) of the male gonad, these EDCs may induce sex-dependent behavioral changes in turtles.     

Endocrine Toxicants and Reproductive Success in Fish

There is compelling evidence on a global scale for compromised growth and reproduction, altered development, and abnormal behaviour in feral fish that can be correlated or in some cases causally linked with exposure to endocrine disrupting chemicals (EDCs). Attributing cause and effect relationships for EDCs is a specific challenge for studies with feral fish as many factors including food availability, disease, competition and loss of habitat also affect reproduction and development. Even in cases where there are physiological responses of fish exposed to EDCs (e.g., changes in reproductive hormone titres, vitellogenin levels), the utility of these measures in extrapolating to whole animal reproductive or developmental outcomes is often limited. Although fish differ from other vertebrates in certain aspects of their endocrinology, there is little evidence that fish are more sensitive to the effects of EDCs. Therefore, to address why endocrine disruption seems so widespread in fish, it is necessary to consider aspects of fish physiology and their environment that may increase their exposure to EDCs. Dependence on aquatic respiration, strategies for iono-osmotic regulation, and maternal transfer of contaminants to eggs creates additional avenues by which fish are exposed to EDCs. This paper provides an overview of responses observed in feral fish populations that have been attributed to EDCs and illustrates many of the factors that need consideration in evaluating the risks posed by these chemicals.     

Adsorption and aerobic biodegradation of four selected endocrine disrupting chemicals in soil–water system

Adsorption and aerobic biodegradation characteristics of four selected endocrine disrupting chemicals (EDCs), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), bisphenol A (BPA) and nonylphenol (NP) were investigated in soil–water system. The sorption of EDCs onto the soil was in the following order: NP > E2 > EE2 > BPA. Sorption isotherms of the four compounds fitted Freundlich models well. The aerobic biodegradation rates of these selected EDCs in the soil–water system could be described by pseudo-first-order kinetic equation. In a single chemical system, the half-lives of EDCs were 1.7, 5.3, 2.7 and 3.3 d for E2, EE2, NP and BPA, respectively, indicating that EE2 was not as readily biodegradable as the others. In a binary-chemical system, the half-lives of EDCs in all cominations were 1.5–2.2 times prolonged than the single chemical system. The following biotransformation pathway of estrogen was proposed: E2/EE2 → E1 → E3. An aerobic conversion of EE2 to E3 was also observed. The result of this research could be useful for predicting environmental fate and ecological risks of EDCs in natural environments especially when soil is their depository.     

Size control of silica nanoparticles and their surface treatment for fabrication of dental nanocomposites

Nearly monodispersed silica nanoparticles having a controlled size from 5 to 450 nm were synthesized via a sol-gel process, and then the optimum conditions for the surface treatment of the synthesized silica nanoparticles with a silane coupling agent (i.e., 3-methacryloxypropyltrimethoxysilane (gamma-MPS)) were explored to produce dental composites exhibiting enhanced adhesion and dispersion of silica nanoparticles in the resin matrix. The particle size was increased by increasing amounts of the catalyst (NH4OH) and silica precursor (tetraethylorthosilicate, TEOS) and by decreasing the amount of water in the reaction mixtures regardless of solvents used for the synthesis. The particle size prepared by using ethanol as a solvent was significantly larger than that prepared by using methanol as a solvent when the composition of the reaction mixture was fixed. The nanosized particles in the 5-25 nm range were aggregated. The amount of grafted gamma-MPS on the surface of the synthesized silica nanoparticles was dependent on the composition of the reaction mixture when an excess amount of gamma-MPS was used. When surface treatment was performed at optimum conditions found here, the amount of the grafted gamma-MPS per unit surface area of the silica nanoparticles was nearly the same regardless of the particle size. Dispersion of the silica particles in the resin matrix and interfacial adhesion between silica particles and resin matrix were enhanced when surface treated silica nanoparticles were used for preparing dental nanocomposites.     

Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.     

Endocrine disruptors: present issues, future directions

A variety of natural products and synthetic chemicals, known collectively as endocrine-disrupting compounds (EDCs), mimic or interfere with the mechanisms that govern vertebrate reproductive development and function. At present, research has focused on (i) the morphological and functional consequences of EDCs; (ii) identifying and determining the relative potencies of synthetic and steroidal compounds that have endocrine-disrupting effects; (iii) the mechanism of action of EDCs at the molecular level; and (iv) the recognition that in "real life," contamination usually reflects mixtures of EDCs. Future research must examine (i) the interactive nature of EDCs, particularly whether the threshold concept as developed in traditional toxicological research applies to these chemicals; (ii) when and how EDCs act at the physiological level, particularly how they may organize the neural substrates of reproductive physiology and behavior; (iii) the various effects these compounds have on different species, individuals, and even tissues; and (iv) how adaptations may evolve in natural populations with continued exposure to EDCs. Several predictions are offered that reflect these new perspectives. Specifically, (i) the threshold assumption will be found not to apply to EDCs because they mimic the actions of endogenous molecules (e.g., estrogen) critical to development; hence, the threshold is automatically exceeded with exposure. (ii) Behavior can compound and magnify the effects of EDCs over successive generations; that is, bioaccumulated EDCs inherited from the mother not only influence the morphological and physiological development of the offspring but also the offsprings' reproductive behavior as adults. This adult behavior, in turn, can have further consequences on the sexual development of their own young. (iii) The sensitivity of a species or an individual to a compound is related to species (individual)-typical concentrations of circulating gonadal steroid hormones. Related to this is the recent finding that alternate forms of the putative receptors are differentially distributed, thereby contributing to the different effects that have been observed. (iv) Except in extraordinary situations, populations often continue to exist in contaminated sites. One possible explanation for this observation that needs to be considered is that animals can rapidly adapt to the nature and level of contamination in their environment. It is unlikely that successive generations coincidentally become insensitive to gonadal steroid hormones fundamentally important as biological regulators of development and reproduction. Rather, adaptive alterations in the genes that encode steroid receptors may occur with chronic exposure to EDCs, allowing the sex hormone receptor to discriminate natural steroids from EDCs.     

Inhibition by wheat sprout (Triticum aestivum) juice of bisphenol A-induced oxidative stress in young women

For health of future generation, fertile young women should be monitored for exposure of endocrine disrupting chemicals (EDCs). Among EDCs, bisphenol A (BPA) is suggested to induce reactive oxygen species (ROS) which play an important role in pathologies of female diseases such as endometriosis. On the other hand, previous studies suggested that sprouts of wheat (Triticum aestivum) have antimutagenicity and antioxidant activity. We performed the 2 weeks intervention of wheat sprout juice (100ml/day) to investigate its effects on BPA-exposure and -oxidative toxicity in young women (N=14, age=24.4±4.0). Geometrical mean of urinary BPA levels was 1.81 (GSTD, 4.34)μg/g creatinine. We observed that irregular meals significantly increased levels of urinary BPA approximate 3 times (p=0.03). In addition, we found BPA-induced oxidative stress is correlated with levels of 8-hydroxydeoxyguanosine (8-OHdG) or malondialdehyde (MDA) levels (p=0.18 or 0.03, respectively). We also observed a continuous reduction of urinary BPA during the wheat sprout intervention (p=0.02). In summary, our data suggested potential detoxification of wheat sprouts on BPA-toxicity via antioxidative and interference of absorption, distribution, metabolism and excretion (ADME)-mediated mechanisms in young women.     

Elimination of Endocrine Disrupting Chemicals using White Rot Fungi and their Lignin Modifying Enzymes: A Review

The ability of white rot fungi (WRF) and their lignin modifying enzymes (LMEs), i.e. laccase and lignin‐ and manganese‐dependent peroxidase, to treat endocrine disrupting chemicals (EDCs) is extensively reviewed in this paper. These chemicals cause adverse health effects by mimicking endogenous hormones in receiving organisms. The alkylphenolic EDCs nonylphenol, bisphenol A and triclosan, the phthalic acid esters dibutylphthalate, diethylphthalate and di‐(2‐ethylhexyl)phthalate, the natural estrogens estrone, 17β‐estradiol, estriol and 17α‐ethynylestradiol and the phytoestrogens genistein and β‐sitosterol have been shown to be eliminated by several fungi and LMEs. WRF have manifested a highly efficient removal of EDCs in aqueous media and soil matrices using both LME and non LME‐systems. The ligninolytic system of WRF could also be used for the elimination of several EDCs and the associated hormone‐mimicking activity. The transformation of EDCs by LMEs and WRF is supported by emerging knowledge on the physiology and biochemistry of these organisms and the biocatalytic properties of their enzymes. Due to field reaction conditions, which drastically differ from laboratory conditions, further efforts will have to be directed towards developing robust and reliable biotechnological processes for the treatment of EDC‐contaminated environmental matrices.