Degradation of ethinyl estradiol by nitrifying activated sludge
Introduction
Increased vitellogenin concentrations in fish recently suggested the presence of biologically active amounts of estrogenic activity in the effluent of sewage treatment plants. It was suggested that amongst others biodegradation products of alkylphenol ethoxylates, and natural and synthetic estrogens might be responsible (Purdom et al., 1994). Of the estrogens naturally synthesised and excreted by women, 17β-estradiol and estrone were detected by GC–MS in effluents of sewage treatment plants. In addition, the synthetic estrogen ethinyl estradiol (EE2), excreted by women using the contraceptive pill, was found Desbrow et al., 1998, Belfroid et al., 1999. The EE2 concentrations found in the effluent of these treatment plants ranged from undetectable levels to 7 ng l−1.
Natural and synthetic estrogens comprise substances with a high pharmacological activity, and although relatively minor amounts of estrogens enter the environment, biodegradation is important to minimise possible biological effects. Many organic compounds are biodegraded by organisms that utilise these compounds for growth; this pathway is probably responsible for the biodegradation of the natural estrogens. Cometabolism – in which an organic compound is modified but not utilised for growth – is another important biodegradation process (Alexander, 1994). Knowledge about the cometabolic transformation of EE2 is essential to assess the fate and potential effects of this compound, as it is known that oxidation of EE2 will lead to a considerable reduction in pharmacological activity (Bergink et al., 1983).
Several bacterial strains that produce monooxygenase enzymes are known to aerobically cometabolise organic compounds. Nitrosomonas europaea is a ubiquitous monooxygenase-producing bacterium catalysing the oxidation of ammonium in soils, natural waters and nitrifying activated sludge. It has often been demonstrated that ammonium monooxygenase (AMO) in the cells of Nitrosomonas europaea is capable of co-oxidising many organic compounds. Examples of co-oxidations include several halogenated hydrocarbons Rashe et al., 1990a, Rashe et al., 1990b, Rashe et al., 1991, aromatics (Keener and Arp, 1994), ethers (Hyman et al., 1994) and thioethers (Hyman et al., 1994). The substrate range of AMO also extends to several hydrocarbons such as alkanes Hyman and Wood, 1983, Hyman et al., 1988 and alkenes (Hyman et al., 1988). Because of the wide metabolic activity of nitrifying bacteria, we sought to test the ability of these organisms to oxidise EE2. In this paper we describe for the first time the capacity of nitrifying sludge to convert EE2.
Section snippets
Chemicals
[3H]-ethinyl estradiol was prepared by reductive tritiation of 2-bromoestrone followed by ethinylation with lithium acetylide ethylenediamine complex. The product was purified by column chromatography, high-performance liquid chromatography (HPLC) and gel filtration. The specific activity was 35 Ci mmol−1 (1.3 TBq mmol−1) as determined by HPLC in combination with liquid scintillation counting. 3H-NMR analysis indicated 70% of the label at C-2, 20% of the label at C-9α and minor amounts of label
Results and discussion
Nitrifying micro-organisms were enriched in a laboratory-scale activated sludge system fed with 4 g l−1 (NH4)2SO4 as the sole energy source. The ammonium oxidation rate of the nitrifying sludge of this enrichment culture was calculated from the disappearance of ammonium over time in batch cultures. The nitrifying activated sludge was capable of oxidising ammonium at a rate of 50 mg NH4 g−1 DW h−1. A representative experiment of EE2 degradation by nitrifying sludge is shown in Fig. 2. This
Acknowledgments
We thank J.P. Sumpter for helpful comments.
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