Removal of estrogenic activity and formation of oxidation products during ozonation of 17alpha-ethinylestradiol

This study investigated the oxidation of the oral contraceptive 17alpha-ethinylestradiol (EE2) during ozonation. First, the effect of ozone (O3) on the estrogenic activity of aqueous solutions of EE2 was studied using a yeast estrogen screen (YES). It could be shown that O3 doses typically applied for the disinfection of drinking waters were sufficient to reduce estrogenicity by a factor of more than 200. However, it proved impossible to completely remove estrogenic activity due to the slow reappearance of 0.1-0.2% of the initial EE2 concentration after ozonation. Second, oxidation products formed during ozonation of EE2 were identified with LC-MS/MS and GC/MS and the help of the model compounds 5,6,7,8-tetrahydro-2-naphthol (THN) and 1-ethinyl-1-cyclohexanol (ECH), which represent the reactive phenolic moiety and the ethinyl group of EE2. Additionally, oxidation products of the natural steroid hormones 17beta-estradiol (E2) and estrone (E1) were identified. The chemical structures of the oxidation products were significantly altered as compared to the parent compounds, explaining the diminished estrogenic activity after ozonation. Overall,the results demonstrate that ozonation is a promising tool for the control of EE2, E2, and E1 in drinking water and wastewater.     

Anaerobic transformation kinetics and mechanism of steroid estrogenic hormones in dairy lagoon water

Wastewater from concentrated animal feeding operations (CAFOs) frequently contains high concentrations of steroid estrogenic hormones. Release of these hormones into the environment may occur when CAFO wastewater is applied to agricultural lands as a nutrient and water source for crop production. To assess the potential risk of hormone contaminants derived from animal wastewater, we investigated the transformation kinetics and mechanisms of three natural estrogenic hormones (17α-estradiol, 17β-estradiol, and estrone) in aqueous solutions blended with dairy lagoon water under anaerobic conditions. Initial transformations of the three hormones in the dairy lagoon water were dominated by biodegradation and the degradation rates were temperature-dependent. The total amounts of hormones (initial concentration at 5 mg L(-1)) remaining in the solution after 52 days at 35 °C accounted for approximately 85%, 78%, and 77% of the initial amounts of 17α-estradiol, 17β-estradiol, and estrone, respectively. This observation suggests that these hormones are relatively stable over time and may accumulate in anaerobic or anoxic environments and anaerobic CAFO lagoons. A racemization reaction between 17α-estradiol and 17β-estradiol via estrone was observed in aqueous solutions in the presence of CAFO wastewater under anaerobic conditions. The initial hormone concentrations did not affect this degradation mechanism. A reversible reaction kinetic model was applied to fit the observed transformation dynamics. The degradation and regeneration of the parent hormone and its metabolites were successfully simulated by this model. The information in this study is useful for assessing the environmental risk of steroid hormones released from CAFO wastewater and to better understand why these hormone contaminants persist in many aquatic environments.     

Functional associations between two estrogen receptors, environmental estrogens, and sexual disruption in the roach (Rutilus rutilus)

Wild male roach (Rutilus rutilus) living in U.K. rivers contaminated with estrogenic effluents from wastewater treatment works show feminized responses and have a reduced reproductive capability, but the chemical causation of sexual disruption in the roach has not been established. Feminized responses were induced in male roach exposed to environmentally relevant concentrations of the pharmaceutical estrogen 17alpha-ethinylestradiol, EE2 (up to 4 ng/ L), during early life (from fertilization to 84 days posthatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ERalpha. and ERbeta, in the brain and gonad/ liver. Transactivation assays were developed for both roach ER subtypes and the estrogenic potencies of steroidal estrogens differed markedly at the different ER subtypes. EE2 was by far the most potent chemical, and estrone (E1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ERbeta was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ERalpha. These findings add substantial support to the hypothesis that steroidal estrogens play a significant role in the induction of intersex in roach populations in U.K. rivers and that the molecular approach described could be usefully applied to understand interspecies sensitivity to xenoestrogens.     

Relative potencies and combination effects of steroidal estrogens in fish

The natural steroids estradiol-17beta (E2) and estrone (E1) and the synthetic steroid ethynylestradiol-17alpha (EE2) have frequently been measured in waters receiving domestic effluents. All of these steroids bind to the estrogen receptor(s) and have been shown to elicit a range of estrogenic responses in fish at environmentally relevant concentrations. At present, however, no relative potency estimates have been derived for either the individual steroidal estrogens or their mixtures in vivo. In this study the estrogenic activity of E2, E1, and EE2, and the combination effects of a mixture of E2 and EE2 (equi-potent fixed-ratio mixture), were assessed using vitellogenin induction in a 14-day in vivo juvenile rainbow trout screening assay. Median effective concentrations, relative to E2, for induction of vitellogenin were determined from the concentration-response curves and the relative estrogenic potencies of each of the test chemicals calculated. Median effective concentrations were between 19 and 26 ng L(-1) for E2, 60 ng L(-1) for E1, and between 0.95 and 1.8 ng L(-1) for EE2, implying that EE2 was approximately 11 to 27 times more potent than E2, while E2 was 2.3 to 3.2 times more potent than E1. The median effective concentration, relative to E2, for the binary mixture of E2 and EE2 was 15 ng L(-1) (comprising 14.4 ng L(-1) E2 and 0.6 ng L(-1) EE2). Using the model of concentration addition it was shown that this activity of the binary mixture could be predicted from the activity of the individual chemicals. The ability of each individual steroid to contribute to the overall effect of a mixture, even at individual no-effect concentrations, combined with the high estrogenic potency of the steroids, particularly the synthetic steroid EE2, emphasizes the need to consider the total estrogenic load of these chemicals in our waterways.     

Sunlight-induced photochemical decay of oxidants in natural waters: implications in ballast water treatment

The transport and discharge of ship ballast water has been recognized as a major vector for the introduction of invasive species. Chemical oxidants, long used in drinking water and wastewater treatment, are alternative treatment methods for the control of invasive species currently being tested for use on ships. One concern when a ballasted vessel arrives in port is the adverse effects of residual oxidant in the treated water. The most common oxidants include chlorine (HOCl/OCl-), bromine (HOBr/OBr-), ozone (03), hydrogen peroxide (H2O2), chlorine dioxide (ClO2), and monochloramine (NH2Cl). The present study was undertaken to evaluate the sunlight-mediated photochemical decomposition of these oxidants. Sunlight photodecomposition was measured at various pH using either distilled water or oligotrophic Gulf Stream water for specific oxidants. For selected oxidants, quantum yields at specific wavelengths were obtained. An environmental photochemical model, GCSOLAR, also provided predictions of the fate (sunlight photolysis half-lives) of HOCI/OCl-, HOBr/OBr-, ClO2, and NH2Cl for two different seasons at latitude 40 degrees and in water with two different concentrations of chromophoric dissolved organic matter. These data are useful in assessing the environmental fate of ballast water treatment oxidants if they were to be discharged in port.     

Unexpected products and reaction mechanisms of the aqueous chlorination of cimetidine

Many pharmaceuticals and personal care products (PPCPs) resist degradation in wastewater treatment plants. Thus, they may be transformed by chemical disinfectants in the final treatment stage, generating products that may possess enhanced toxicity/biological activity relative to the parent compounds. For this reason, the reaction of cimetidine, an over-the-counter antacid, with the frequently used disinfectant, free chlorine, was investigated. Cimetidine degraded rapidly in the presence of excess free chlorine, indicating that it will likely undergo significant transformation during wastewater disinfection. Four major products were isolated and extensively characterized by comparison of liquid chromatographic retention times to known standards, mass spectrometry, 1H- and 2D-nuclear magnetic resonance spectroscopy, and infrared spectroscopy. An expected sulfur oxidation product, cimetidine sulfoxide, was identified along with three unexpected products: 4-hydroxymethyl-5-methyl-1H-imidazole, 4-chloro-5-methyl-1H-imidazole, and a product proposed to be either a beta- or delta-sultam. The last three products are formed by transformations not frequently observed in free chlorine reactions of PPCPs such as C-C bond cleavage and intramolecular nucleophilic substitution. The unexpected transformations yielded compounds with more substantial structural changes than would be observed in common free chlorine reactions such as N-chlorination or electrophilic halogenation. The reaction pathway was elucidated by kinetic analysis and by independently treating isolated intermediates with free chlorine, and reaction mechanisms were proposed. Finally, the predicted no-effect concentration (PNEC) of the chlorination products was estimated, and the products 4-hydroxymethyl-5-methyl-1H-imidazole and 4-chloro-5-methyl-1H-imidazole were estimated to have lower PNECs than cimetidine.     

Estrogenic activity and steroid hormones in swine wastewater through a lagoon constructed-wetland system

Anaerobic lagoons and treatment wetlands are used worldwide to treat wastewater from dense livestock production facilities; however, there is very limited data on the hormonal activity of the wastewater effluent produced by these treatment systems. The objectives of this experiment were to measure (1) the hormonal activity of the initial effluent and (2) the effectiveness of a lagoon-constructed wetland treatment system for producing an effluent with a low hormonal activity. Wastewater samples were taken in April, July, and November 2004 and July 2005 from a lagoon-constructed wetland system at a swine farrowing facility. Estrogenic activity (in vitro E-screen assay), 17 beta-estradiol (E2), and testosterone concentrations (LC/MS-MS) were measured. A high correlation was found between estradiol equivalents determined by E-screen and LC/MS-MS (R2 = 0.82). Nutrient removal was measured to ensure that the wetlands were functioning in a manner similar to literature reports. Nutrient removals were typical for treatment wetlands: TKN 59-75% and orthophosphate 0-18%. Wetlands decreased estrogenic activity by 83-93%. Estrone was the most persistent estrogenic compound. Constructed wetlands produced effluents with estrogenic activity below the lowest equivalent E2 concentration known to have an effect on fish (10 ng/L or approximately 37 x 10(-12) M).     

17alpha-ethinylestradiol transformation via abiotic nitration in the presence of ammonia oxidizing bacteria

Impacts of trace concentrations of estrogens on aquatic ecosystems are a serious environmental concern, with their primary source being wastewater treatment facility effluents. Increased removal of 17alpha-ethinylestradiol (EE2) has been reported for activated sludge treatment with long enough solids retention time for nitrification. Previous work based on batch tests with Nitrosomonas europaea and nitrifying activated sludge at high EE2 concentrations (>300 000 ng/L) and high NH4-N concentrations (>200 mg/L) has led to the hypothesis that ammonia oxidizing bacteria cometabolically degrade EE2. This work investigated EE2 transformation with N. europaea and Nitrosospira multiformis at environmentally relevant EE2 concentrations and LC-MS-MS to observe transformation products. Degradation of EE2 was not observed in batch tests with no NH4-N addition or with 10 mg/L NH4-N fed daily. At increased NH4-N concentrations (200-500 mg/L) EE2 transformation was observed, but the only detected products were nitrated EE2. Abiotic assays with growth medium confirmed EE2 removal by nitration, which is enhanced at low pH and high NO2-N concentrations. These results suggest that EE2 removal at low concentrations found in municipal treatment activated sludge systems is not due to cometabolic degradation by ammonia oxidizing bacteria, or to abiotic nitration, but most likely due to heterotrophic bacteria.     

Destruction of estrogens using Fe-TAML/peroxide catalysis

Endocrine disrupting chemicals (EDCs) impair living organisms by interfering with hormonal processes controlling cellular development Reduction of EDCs in water by an environmentally benign method is an important green chemistry goal. One EDC, 17alpha-ethinylestradiol (EE2), the active ingredient in the birth control pill, is excreted by humans to produce a major source of artificial environmental estrogenicity, which is incompletely removed by currenttechnologies used by municipal wastewater treatment plants (MWTPs). Natural estrogens found in animal waste from concentrated animal feeding operations (CAFOs) can also increase estrogenic activity of surface waters. An iron-tetraamidomacrocyclic ligand (Fe-TAML) activator in trace concentrations activates hydrogen peroxide and was shown to rapidly degrade these natural and synthetic reproductive hormones found in agricultural and municipal effluent streams. On the basis of liquid chromatography tandem mass spectrometry, apparent half-lives for 17 alpha- and 17 beta-estradiol, estriol, estrone, and EE2 in the presence of Fe-TAML and hydrogen peroxide were approximately 5 min and included a concomitant loss of estrogenic activity as established by E-Screen assay.     

Occurrence of estrogenic compounds in and removal by a swine farm waste treatment plant

The total estrogenic activity of the wastewater from a swine farm in Japan was quantitatively characterized, and the compounds responsible for the estrogenic activity were identified and quantified. The wastewater treatment process consisted of a series of an up-flow anaerobic sludge blanket (UASB) and a trickling filter. Samples were collected at each treatment step, and the total estrogenic activity was determined by use of an in vitro gene expression assay (MVLN; MCF-7 human breast cancer cell stably transfected with the pVit-tk-LUC receptor plasmid). Individual estrogenic compounds were identified and quantified using liquid chromatography-mass spectrometry (LC/MS) and liquid chromatography-tandem mass spectrometry (LC/ MS/MS). To further identify the compounds contributing to the estrogenic activity in the wastewater, the sample extracts were fractionated into 12 fractions (fractions 1-12) by HPLC. The rate of removal of estrogenic activity between the effluent and the influent was greater than 97%. The trickling filter removed the majority of the estrogenic activity. The removal rates of specific estrogenic compounds ranged from 44 to 99%. Estrogenic activity was detected mainly in the fractions containing estrone (El), 17beta-estradiol (betaE2), 17alpha-estradiol (alpha E2), estriol (E3), bisphenol A (alphaPA), and equol (EQ0). The ratios of betaE2-EQc (betaE2 equivalents derived from chemical analysis) to betaE2-EQB (betaE2 equivalent derived from bioassay) in the 12 fractions collectively were contributed by El (17-30%), betaE2 (23-30%), acE2 (<1%), E3 (1-2%), BPA (<1%), and EQO (2-3%) in the influent and El (16-37%), PE2 (<1-7%), alphaE2 (<1%), E3 (<1-3%), BPA (<1%), and EQO (<1%) in the effluent. The compounds responsible for most of the estrogenic activity measured in the bioassay were natural estrogens such as El and betaE2.     

Assessment of the reproductive-endocrine disrupting potential of chlorine dioxide oxidation products of plant sterols

This study examined the hypothesis that chlorine dioxide bleaching used in pulp and paper production causes the formation of reproductive-endocrine disrupting compounds from plant sterols. This was tested by conducting a laboratory simulation of the chlorine dioxide oxidation of two plant sterols, beta-sitosterol and stigmasterol. Oxidation products of the plant sterol beta-sitosterol were purified and identified and found to be cholestan-24-ethyl-3-one, 4-cholestene-24-ethyl-3-one, and 4-cholestene-24-ethyl-3,6-dione. The first two compounds were found in a number of pulp and paper effluents and biosolids. The sterols and their oxidation products were tested in vitro using bioassays for androgenicity and estrogenicity. A 28 d in vivo bioassay was employed to examine masculinization in female mosquitofish. In vitro bioassays revealed little estrogenic activity in the parent sterols or in mixtures of their oxidation products. Androgenic activity as measured by the androgen receptor binding bioassay was in the order of 19-96 microg/g testosterone equivalents but with no increase or decrease with chlorine dioxide oxidation. The mosquitofish bioassay did not show significant masculinization for any of the preparations tested. A number of androstane steroids were identified in the sterols tested, however, those compounds could only account for a small fraction of the androgenic activity in the sterols. It was clear that the parent sterols were not themselves acting as androgens in the bioassays used. This study indicated that chlorine dioxide oxidation of sterols produced predominantly oxidized sterols that were not likely to act through androgenic or estrogenic mechanisms.     

Comparison of in vivo and in vitro reporter gene assays for short-term screening of estrogenic activity

Functional in vitro and in vivo reporter gene assays have recently been developed for the rapid determination of exposure to (xeno)estrogens. The in vitro estrogen receptor (ER)-mediated chemically activated luciferase gene expression (ER-CALUX) assay uses T47D human breast cancer cells stably transfected with an ER-mediated luciferase gene construct. In the in vivo assay, transgenic zebrafish are used in which the same luciferase construct has been stably introduced. In both assays, luciferase reporter gene activity can be easily quantified following short-term exposure to chemicals activating endogenous estrogen receptors. The objective of this study was to compare responses by known (xeno)estrogenic compounds in both assays. Exposure to the (xeno)estrogens estradiol (E2), estrone, ethynylestradiol (EE2), o,p'-DDT, nonylphenol (NP), and di(2-ethylhexyl)phthalate (DEHP) revealed that EE2 was the most potent (xeno)estrogen tested and was 100 times more potent than E2 in the transgenic zebrafish assay, whereas in the in vitro ER-CALUX assay, EE2 and E2 were equipotent Although the xenoestrogens o,p'-DDT and NP were full estrogen agonists in the in vitro ER-CALUX assay, only o,p'-DDT demonstrated weak dose-related estrogenic activity in vivo. To determine if differences in reporter gene activity may be explained by differential affinity of (xeno)estrogens to human and zebrafish ERs, full-length sequences of the zebrafish ER subtypes alpha, beta, and gamma were cloned, and transactivation by (xeno)estrogens was compared to human ERalpha and ERbeta. Using transiently transfected recombinant ER and reporter gene constructs, EE2 also showed relatively potent activation of zebrafish ERalpha and ERbeta compared to human ERalpha and ERbeta. Zebrafish ERbeta and ERgamma showed higher transactivation by (xeno)estrogens relative to E2 than human ERbeta.     

FeCl3/NaNO2: an efficient photocatalyst for the degradation of aquatic steroid estrogens under natural light irradiation

Assistance and acceleration of the environment's self-remediation of pollutants represent an important and long-standing goal for environmental chemistry communities. Here, a degradation route using a combination of a nitrite and a ferric salt as the photocatalyst is presented for catalytically removing 17beta-estradiol (E2), estriol (E3), and 17alpha-ethynylestradiol (EE2) in water under mimicked natural environmental conditions, i.e., in the phytotron. After a 1 day reaction, 86.6% of the estrogen E2 was degraded. Extending the incubation time to 30 days, more than 99.9% E2 was removed and a very small quantity of malonic acid observed as the residual organic compound, and estrogenic activity was determined. The results showed that the estrogenic activities of the intermediate products are negligible and that there is no secondary risk associated with increased the estrogenic activity. The degradation system demonstrated that FeCl3/NaN02 is an efficient photocatalyst which is active on natural light irradiation. This work highlights a promising development for in situ treatment of pollutants in natural-environment conditions.     

Estrogen receptor agonist fate during wastewater and biosolids treatment processes: a mass balance analysis

The estrogen receptor agonist fate of hexane extracts from various locations and phases (liquid and solid) within one pilot-scale and two full-scale wastewater treatment facilities were examined by use of the receptor-binding yeast estrogen screen (YES assay). Estrogenic activity was found in samples that contained a high concentration of biological solids and was particularly high in the suspended solid fraction from biosolids treatment facilities. Mass balances revealed that the estrogenic activity associated with the processed biosolids constituted between 5 and 10% of the influent estrogenic activity, while the treated liquid effluent prior to disinfection contained between 26 and 43%. Overall, this suggests that between 51 and 67% of the estrogenic activity contained in the influent wastewater was either biodegraded during the wastewater or biosolids treatment processes or was unavailable to the extraction/detection procedure. In both aerobic and anaerobic digestion, mass balances revealed an increase in estrogenic activity as treatment progressed and biosolids destruction occurred. The estrogenic activity associated with the solid phase decreased during mesophilic aerobic digestion. A correlation was observed between the estrogenicity of mixed liquor suspended solids and aerobic sludge age and suggests that wastewater treatment facilities can be designed and operated to enhance the sorption and removal of estrogenic compounds from the liquid phase.     

Production of macromolecular chloramines by chlorine-transfer reactions

Chlorination of treated wastewaters is undertaken to prevent dispersal of human pathogens into the environment. Except in well-nitrified effluents, the primary agents in chlorination, Cl2(g) or NaOCl(aq), are short-lived and quickly transfer oxidative chlorine to secondary agents (N-chloramines), which then participate in the disinfection process. Maturation of residual chlorine resulting from chlorine-transfer reactions is still poorly characterized. Using gel permeation and reversed-phase liquid chromatography combined with a novel, oxidant-specific detector, unanticipated trends during the maturation of residual chlorine in wastewater are identified. Within 2 min after addition of NaOCl, and continuing for several hours at least, significant amounts of oxidative chlorine are transferred to secondary agents that are moderately to strongly hydrophobic and to agents that have high relative molecular masses (Mr 1300-25000). It is hypothesized that hydrophobic stabilization of organic chloramines (RNHCl(o)) thermodynamically drives these transfers, making macromolecular chloramines the ultimate oxidative chlorine carriers. Macromolecular chloramines are expected to be sluggish oxidants, as observed in their reduction by sulfite, and are expected to be poor disinfectants. If transfer of oxidative chlorine to high Mr components occurs widely at treatment plants, then this phenomenon offers a new, physicochemical explanation for the well-known impotency of organic chloramines in wastewater disinfection.     

Gene expression profiling for understanding chemical causation of biological effects for complex mixtures: a case study on estrogens

Gene expression profiling offers considerable potential for identifying chemical causation of effects induced in exposures to complex mixtures, and for understanding the mechanistic basis for their phenotypic effects. We characterized gene expression responses in livers and gonads of fathead minnow (Pimephales promelas) exposed (for 14-21 days) to estrogenic wastewater treatment works final effluents with varying potencies and assessed the extent to which these expression profiles mapped with those induced by individual steroid estrogens present in the effluents (17beta-estradiol and 17alpha-ethinylestradiol) and, thus, were diagnostic of estrogen exposure. For these studies, we adopted a targeted approach (via real-time PCR) with a suite of 12 genes in liver and 21 genes in gonad known to play key roles in reproduction, growth and development (processes controlled by estrogens) and responses were compared with effects on phenotypic end points indicative of feminization. Gene responses to effluent were induced predominantly in a linear (monotonic) concentration-dependent manner but were complex with many genes responding differently between tissue types and sexes. The gene expression profiles for the estrogenic effluents and the individual steroid estrogens had many common features. There were marked differences in the profiles between the two effluents, however, that were not explained by differences in their estrogenic potencies, suggesting that these may have arisen as a consequence of differences in the contents of other chemicals, which may act directly or indirectly with the estrogen-response pathway to alter estrogen-induced gene expression. These data demonstrate that the patterns of gene expression induced by estrogenic effluents, although complex, can be diagnostic for some of the estrogens they contain and provide insights into the mechanistic basis for the phenotypic effects seen.     

17 beta-estradiol degradation by TiO2 photocatalysis as a means of reducing estrogenic activity

The degradation of 17 beta-estradiol (E2) in water by TiO2 photocatalysis was investigated; concurrently the estrogenic activity of the treated water was evaluated during the photocatalytic reactions by an estrogen screening assay. As a result, 10(-6) M of E2 was totally mineralized to CO2 in 1.0 g L-1 TiO2 suspension under UV irradiation for 3 h. 10 epsilon-17 beta-Dihydroxy-1,4-estradien-3-one and testosterone-like species were elucidated as intermediate products by GC/MS analysis. The mechanisms of E2 degradation by TiO2 photocatalysis were discussed not only experimentally but also theoretically by calculating the frontier electron densities of the E2 molecule. On the basis of the results obtained, it was concluded that the phenol moiety of the E2 molecule, one of the essential functional groups to interact with the estrogen receptor, should be the starting point of the photocatalytic oxidation of E2. This means that the estrogenic activity should be almost lost concurrently with the initiation of the photocatalytic degradation. Actually, the estrogenic activities of the intermediate products were negligible. TiO2 photocatalysis could be applied to water treatment to effectively remove natural and synthetic estrogens without producing biologically active intermediary products.     

Using electronic theory to identify metabolites present in 17α-ethinylestradiol biotransformation pathways

This research used electronic theory to model the biotransformation of 17α-ethinylestradiol (EE(2)) under aerobic conditions in mixed culture. The methodology involved determining the Frontier Electron Density (FED) for EE(2) and various metabolites, as well as invoking well-established degradation rules to predict transformation pathways. We show that measured EE(2) metabolites are in good agreement with what is expected based on FED-based modeling. Initiating reactions occur at Ring A, producing metabolites that have been experimentally detected. When OH-EE(2) and 6AH-EE(2) are transformed, Ring A is cleaved before Ring B. The metabolites involved in these pathways have different estrogenic potentials, as implied by our analysis of the log P values and the hydrogen bonding characteristics. The OH-EE(2) and 6AH-EE(2) transformation pathways also show redox-induced electron rearrangement (RIER), where oxidation reactions lead to the reduction of carbon units present along the bond axis. Sulfo-EE(2) appears to be difficult to biotransform. These findings clarify theoretical and practical aspects of EE(2) biotransformation.