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Degradation of ethinyl estradiol by nitrifying activated sludge
Chemosphere 41 (2000) 1239±1243
Degradation of ethinyl estradiol by nitrifying activated sludge J.S. Vader a, C.G. van Ginkel b,*, F.M.G.M. Sperling a, J. de Jong a, W. de Boer a, J.S. de Graaf a, M. van der Most c, P.G.W. Stokman a b
a NV Organon, P.O. Box 20, 5340 BH Oss, Netherlands Akzo Nobel Chemical Research BV, P.O. Box 9300, 6800 SB Arnhem, Netherlands c Diosynth BV, P.O. Box 20, 5340 BH Oss, Netherlands
Received 4 August 1999; accepted 17 November 1999
Abstract Degradation of ethinyl estradiol (EE2 ) by nitrifying activated sludge was studied with micro-organisms grown in a reactor with feedback of sludge fed with only a mineral salts medium containing ammonium as the sole energy source. ÿ1 Ammonium was oxidised by this sludge at a rate of 50 mg NH DW hÿ1 . This activated sludge was also capable of 4 g ÿ1 ÿ1 degrading EE2 at a maximum rate of 1 lg g DW h . Using sludge with an insigni®cant nitrifying capacity of 1 mg ÿ1 NH DW hÿ1 , no degradation of EE2 was detected. Oxidation of EE2 by nitrifying sludge resulted in the formation 4 g of hydrophilic compounds, which were not further identi®ed. Most probably degradation by nitrifying sludge results in a loss of estrogenic activity, as hydroxylated derivatives of EE2 are known to have a substantially lower pharmacological activity than EE2 . Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cometabolism; Nitri®cation; Wastewater treatment; Estrogen
1. Introduction Increased vitellogenin concentrations in ®sh recently suggested the presence of biologically active amounts of estrogenic activity in the euent 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, 17b-estradiol and estrone were detected by GC±MS in euents of sewage treatment plants. In addition, the synthetic estrogen ethinyl estradiol (EE2 ), excreted by women using the contraceptive
* Corresponding author. Tel.: +31-26-366-2634; fax: +31-26366-2528. E-mail address: [email protected] (C.G. van Ginkel).
pill, was found (Desbrow et al., 1998; Belfroid et al., 1999). The EE2 concentrations found in the euent of these treatment plants ranged from undetectable levels to 7 ng lÿ1 . 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 eects. 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 modi®ed 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 eects of this compound, as it is known that oxidation of EE2 will lead to a considerable reduction in pharmacological activity (Bergink et al., 1983).
0045-6535/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 5 5 6 - 1
1240
J.S. Vader et al. / Chemosphere 41 (2000) 1239±1243
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,b; 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 ®rst time the capacity of nitrifying sludge to convert EE2 . 2. Materials and methods 2.1. Chemicals [3 H]-ethinyl estradiol was prepared by reductive tritiation of 2-bromoestrone followed by ethinylation with lithium acetylide ethylenediamine complex. The product was puri®ed by column chromatography, high-performance liquid chromatography (HPLC) and gel ®ltration. The speci®c activity was 35 Ci mmolÿ1 (1.3 TBq mmolÿ1 ) as determined by HPLC in combination with liquid scintillation counting. 3 H-NMR analysis indicated 70% of the label at C-2, 20% of the label at C-9a and minor amounts of label at C-6a/b, C-11a and C-12a. 2.2. Activated sludge Activated sludge with an activity of only 1 mg NH4 gÿ1 DW hÿ1 was collected at a wastewater treatment plant in Duiven, the Netherlands. This plant primarily treated domestic wastewater. Nitrifying activated sludge was cultivated in a glass-constructed reactor with feedback of biomass (Fig. 1). The reactor consisted of an aeration vessel (C) with a capacity of 0.36 l, from which the liquor was passed continuously to a settler (D) with a capacity of 0.08 l. The reactor was started by ®lling the system with activated sludge. Euent that left the apparatus was collected in a container (E). Aeration was achieved with an approximate air¯ow of 10 l hÿ1 through a capillary leading to the bottom of the aeration vessel. The nitrifying sludge was grown in a mineral salts medium: 4 g lÿ1 (NH4 )2 SO4 ; 0.2 g lÿ1 MgSO4 7H2 O, 1.5 g lÿ1 K2 HPO4 0.08 g lÿ1 CaCl2 and 0.1 ml lÿ1 of a
Fig. 1. Schematic diagram of the continuously fed activated sludge (CAS) reactor used for the laboratory-scale tests. The unit consists of a storage vessel (A), dosing pump (B), aeration section (C), settling section (D), collecting vessel (E), and air supply (F).
trace solution (Vishniac and Santer, 1957). The ¯ow of the mineral salts medium through the reactor was maintained by using a peristaltic pump at a ¯ow rate of 250 ml dÿ1 . Daily, 18 ml of sludge was removed from the aeration basin to maintain a sludge retention time of 20 days. The concentration of dissolved oxygen was between 2 and 7 mg lÿ1 . The pH in the unit was controlled at 7.5 by automatic titration with 20 g lÿ1 NaHCO3 . The sludge was incubated in diuse light at 20°C. 2.3. Analyses The EE2 content was analysed using HPLC. The HPLC consisted of a pump (Waters 510), a Novapak C18 (3:9 150 mm2 ) column and a UV detector (Separation 785A; LSC detector (bRAM)). The concentration of EE2 was measured with radiochromatography. The mobile phase was acetonitrile/water (40/60, v/v). The ¯ow rate was 1 ml minÿ1 . Samples (50 ll) were injected after sedimentation of the sludge. Ammonium was determined by forming a greencoloured indophenol with salicylate and hypochlorite in an alkaline medium. The absorbance of the complex formed was measured spectrophotometrically at 580 nm. The dry weight (DW) of the sludge was determined by ®ltering a 10 ml sample through a preweighed 12 lm cellulose nitrate membrane ®lter and drying this ®lter at 104°C to a constant weight. 2.4. Batch experiments Activated sludge was withdrawn from the continuously fed reactor or the full-scale reactor and injected into Erlenmeyer ¯asks. Approximately 0.1 mCi (3.7 MBq) [3 H]-ethinyl estradiol in 0.1 ml ethanol was added to the 15 ml sludge in an Erlenmeyer ¯ask. The initial cell density was 1.0 g lÿ1 and the initial EE2
J.S. Vader et al. / Chemosphere 41 (2000) 1239±1243
concentrations were approximately 50 lg ÿ1 l. To provide an exogenous source of energy hydrazine was added to the Erlenmeyer ¯asks at a concentration of 10 mg lÿ1 . Erlenmeyer ¯asks were incubated on a magnetic shaker at 100 rpm and aliquots of 1 ml were taken over time. Aliquots withdrawn were analysed by liquid scintillation counting and HPLC pro®ling. Erlenmeyers containing 0.2 g lÿ1 of nitrifying sludge were shaken at 100 rpm at 20°C for 4 h, to allow almost complete oxidation of the 50 mg ÿ1 l ammonium added. Samples from the Erlenmeyer ¯asks were centrifuged at 5.000 g for 5 min to remove sludge particles prior to making analyses of ammonium.
3. Results and discussion Nitrifying micro-organisms were enriched in a laboratory-scale activated sludge system fed with 4 g lÿ1 (NH4 )2 SO4 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 gÿ1 DW hÿ1 . A representative experiment of EE2 degradation by nitrifying sludge is shown in Fig. 2. This nitrifying sludge degraded EE2 completely within six days and did not require an adaptation period. The initial speci®c EE2 degradation rate by nitrifying sludge was 1 lg gÿ1 DW hÿ1 . Nitrifying activated sludge retained its maximum EE2 degradation abilities for approximately two days. After this period the rate of degradation levelled o, probably due to the anity of the micro-organisms for EE2 at low concentrations in the experiment or to a decrease in the activity of the non-growing cells (Fig. 2). The EE2 biodegradation was con®rmed in two similar experiments. No detectable degradation of EE2 present at a concentration of 50 lg lÿ1 was measured with activated sludge capable
Fig. 2. Degradation of EE2 by nitrifying sludge.
1241
of oxidising ammonium at a rate of only 1 mg NH4 gÿ1 DW hÿ1 . Nitrifying sludge was capable of degrading EE2 in the absence of ammonium. Nitrifying bacteria like Nitrosomonas use ammonium as an energy source and oxidise ammonium to nitrite. As oxidation of ammonium by a monooxygenase requires a source of reducing energy (NADH), the capacity to transform EE2 by the sludge may become a function of the available reducing energy. When hydrazine (10 mg lÿ1 ) was added as an external electron donor for NADH generation, it removed the reducing energy limitation. Nitrifying sludge supplied with hydrazine exhibited slightly higher EE2 degradation rates than sludge without hydrazine. This result indicates that EE2 degradation is mediated by ammonium monooxygenase activity. Ammonium-oxidising bacteria are able to co-metabolise many low molecular weight organic compounds (Hyman and Wood, 1983; Hyman et al., 1988; Rashe et al., 1990a,b; 1991; Juliette et al., 1993; Hyman et al., 1994; Keener and Arp, 1994). Our results demonstrate that nitrifying bacteria not only acts on low molecular weight organic compounds but also on a synthetic steroid. Although the removal of the parent compound already demonstrates the biological degradation of EE2 . the oxidation of EE2 was con®rmed by the formation of hydrophilic organic compounds, as can be seen from early eluting peaks in the radioactivity pro®le (Fig. 3). The pathway of ammonium oxidation is initiated by the enzyme ammonium monooxygenase. Monooxygenases can carry out reactions which insert oxygen into C±H bonds. Ammonium monooxygenase catalyses, for instance, the hydroxylation of alkanes to produce primary and secondary alcohols (Hyman and Wood, 1983; Hyman et al., 1988). The activity of nitrifying activated sludge probably results in hydroxylation, converting EE2 into hydrophilic products. A portion of the very polar radioactivity detected in the radio HPLC was quite volatile (probably tritiated water), indicating extensive hydroxylation of EE2 by the nitrifying activated sludge. These hydroxylated degradation products are essentially devoid of estrogenic activity (Bergink et al., 1983). Nitrogen removal in biological treatment systems is achieved by nitri®cation of all nitrogen to nitrate followed by removal of nitrate through denitri®cation. At present limited data of monitoring studies are available relating the nitrifying activity to the removal of EE2 in wastewater treatment plants. The importance of such data is illustrated by monitoring studies performed by Desbrow et al. (1998) and Belfroid et al. (1999) showing remarkable seasonal changes with respect to EE2 removal. In summer, the content of EE2 in the euents was below the detection limit of 0.2 ng lÿ1 in all four plants situated in England (only one sample of one plant contained an exceptionally high EE2 concentration). In
1242
J.S. Vader et al. / Chemosphere 41 (2000) 1239±1243
Fig. 3. HPLC chromatograms showing the degradation of EE2 and formation of hydrophilic products by nitrifying sludge. HPLC pro®les were from samples taken at the start (above) and the end (below) of a batch experiment. EE2 elutes at 8.5 min.
winter, EE2 concentrations detected ranged from 0.2 to 4.3 ng lÿ1 in two plants. The euent of a third plant monitored in winter contained no EE2 . Comparable seasonal changes were found in treatment plants in the Netherlands (Belfroid et al., 1999). Reduced nitri®cation in biological treatment systems at low temperatures due to wash-out of nitrifying bacteria is a well-known phenomenon. An adequate sludge retention time in relation to the growth rate ensures that the nitrifying bacteria do not wash out, which means that nitri®cation will take place. The higher growth rate of nitrifying bacteria in summer due to increased temperatures enables nitrifying bacteria to maintain themselves at lower sludge retention times. The results of the monitoring studies indicate that the ability to degrade EE2 is correlated with a temperature-dependent microbial processes such as nitri®cation. Although partial removal of ammonium during the winter period in the plant in the Netherlands (Belfroid, personal communication) seems to contradict this correlation, future monitoring studies of waste water treatment plants should record data on the capacity to nitrify and the sludge retention time. Nitrifying bacteria not only occur in biological wastewater treatment systems but also abundantly in natural ecosystems. The process of nitri®cation is considered to be a crucial process in the nitrogen cycle in natural ecosystems such as soils. EE2 degradation by nitrifying micro-organisms is a cometabolic process.
Cometabolic conversions are thought to play a signi®cant role in the degradation of xenobiotic compounds in nature (Alexander, 1994). In summary, nitrifying bacteria are widespread in the environment. As they can degrade EE2 without prior adaptation, it can be concluded that these bacteria represent a sink for EE2 in various environmental compartments and activated sludge systems. Acknowledgements We thank J.P. Sumpter for helpful comments. References Alexander, M., 1994. Biodegradation and bioremediation. Academic Press, San Diego, USA, pp. 177±195. Belfroid, A.C., van der Horst, A., Vethaak, A.D., Sch afer, A.J., Rijs, G.B.J., Wegener, J., Co®no, W.P., 1999. Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in the Netherlands. Sci. Total Environ. 225, 101±108. Bergink, E.W., Kloosterboer, H.J., van der Velden, W.H.M., van der Vlies, J., Winter, M.S., 1983. Speci®city of an estrogen binding protein in the human vagina compared with that of estrogen receptors in dierent tissues from dierent species. In: Jasonni, V.M. (Ed.), Steroids and
J.S. Vader et al. / Chemosphere 41 (2000) 1239±1243 Endometrial Cancer. Raven Press, New York, USA, pp. 77± 84. Desbrow, C., Routledge, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M.J., 1998. Identi®cation of estrogenic chemicals in STW euent 1 Chemical fractionation and in vitro biological screening. J. Environ. Sci. Technol. 32, 1549±1558. Hyman, M.R., Murton, I.B., Arp, D.J., 1988. Interaction of ammonia monooxygenase from Nitrosomonas europeae with alkanes, alkenes and alkynes. Appl. Environ. Microbiol. 54, 3187±3190. Hyman, M.R., Page, C.L., Arp, D.J., 1994. Oxidation of methyl ¯uoride and dimethylether by ammonia monooxygenase in Nitrosomonas europaea. Appl. Environ. Microbiol. 60, 3033±3035. Hyman, M.R., Wood, P.M., 1983. Methane oxidation by Nitrosomonas europaea. Biochem. J. 212, 31±37. Juliette, L.Y., Hyman, M.R., Arp, D.J., 1993. Inhibition of ammonia oxidation in Nitrosomonas europaea by sulfur compound-thioethers are oxidized to sulfoxides by ammonium mono-oxygenase. Appl. Environ. Microbiol. 59, 3718± 3727.
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Keener, W.K., Arp, D.J., 1994. Transformations of aromatic compounds by Nitrosomonas europea. Appl. Environ. Microbiol. 60, 1921±1941. Purdom, C.E., Hardiman, P.A., Bye, V.J., Eno, N.C., Tyler, C.R., Sumpter, J.P., 1994. Estrogenic eects of euents from sewage treatment works. Chemistry and Ecology 8, 275±285. Rashe, M.E., Hicks, R.E., Hyman, M.R., Arp, D.J., 1990a. Oxidation of monohalogenated ethanes and n-chlorinated alkanes by whole cells of Nitrosomonas europae. J. Bacteriol. 172, 5368±5373. Rashe, M.E., Hyman, M.R., Arp, D.J., 1990b. Biodegradation of halogenated hydrocarbon fumigants by nitrifying bacteria. Appl. Environ. Microbiol. 56, 258±2571. Rashe, M.E., Hyman, M.R., Arp, D.J., 1991. Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia mono-oxygenase and substrate speci®city. Appl. Environ. Microbiol. 57, 2986±2994. Vishniac, W., Santer, M., 1957. The Thiobacilli. Bacteriol. Rev. 21, 195±213.
Degradation of ethinyl estradiol by nitrifying activated sludge J.S. Vader a, C.G. van Ginkel b,*, F.M.G.M. Sperling a, J. de Jong a, W. de Boer a, J.S. de Graaf a, M. van der Most c, P.G.W. Stokman a b
a NV Organon, P.O. Box 20, 5340 BH Oss, Netherlands Akzo Nobel Chemical Research BV, P.O. Box 9300, 6800 SB Arnhem, Netherlands c Diosynth BV, P.O. Box 20, 5340 BH Oss, Netherlands
Received 4 August 1999; accepted 17 November 1999
Abstract Degradation of ethinyl estradiol (EE2 ) by nitrifying activated sludge was studied with micro-organisms grown in a reactor with feedback of sludge fed with only a mineral salts medium containing ammonium as the sole energy source. ÿ1 Ammonium was oxidised by this sludge at a rate of 50 mg NH DW hÿ1 . This activated sludge was also capable of 4 g ÿ1 ÿ1 degrading EE2 at a maximum rate of 1 lg g DW h . Using sludge with an insigni®cant nitrifying capacity of 1 mg ÿ1 NH DW hÿ1 , no degradation of EE2 was detected. Oxidation of EE2 by nitrifying sludge resulted in the formation 4 g of hydrophilic compounds, which were not further identi®ed. Most probably degradation by nitrifying sludge results in a loss of estrogenic activity, as hydroxylated derivatives of EE2 are known to have a substantially lower pharmacological activity than EE2 . Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cometabolism; Nitri®cation; Wastewater treatment; Estrogen
1. Introduction Increased vitellogenin concentrations in ®sh recently suggested the presence of biologically active amounts of estrogenic activity in the euent 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, 17b-estradiol and estrone were detected by GC±MS in euents of sewage treatment plants. In addition, the synthetic estrogen ethinyl estradiol (EE2 ), excreted by women using the contraceptive
* Corresponding author. Tel.: +31-26-366-2634; fax: +31-26366-2528. E-mail address: [email protected] (C.G. van Ginkel).
pill, was found (Desbrow et al., 1998; Belfroid et al., 1999). The EE2 concentrations found in the euent of these treatment plants ranged from undetectable levels to 7 ng lÿ1 . 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 eects. 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 modi®ed 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 eects of this compound, as it is known that oxidation of EE2 will lead to a considerable reduction in pharmacological activity (Bergink et al., 1983).
0045-6535/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 5 5 6 - 1
1240
J.S. Vader et al. / Chemosphere 41 (2000) 1239±1243
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,b; 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 ®rst time the capacity of nitrifying sludge to convert EE2 . 2. Materials and methods 2.1. Chemicals [3 H]-ethinyl estradiol was prepared by reductive tritiation of 2-bromoestrone followed by ethinylation with lithium acetylide ethylenediamine complex. The product was puri®ed by column chromatography, high-performance liquid chromatography (HPLC) and gel ®ltration. The speci®c activity was 35 Ci mmolÿ1 (1.3 TBq mmolÿ1 ) as determined by HPLC in combination with liquid scintillation counting. 3 H-NMR analysis indicated 70% of the label at C-2, 20% of the label at C-9a and minor amounts of label at C-6a/b, C-11a and C-12a. 2.2. Activated sludge Activated sludge with an activity of only 1 mg NH4 gÿ1 DW hÿ1 was collected at a wastewater treatment plant in Duiven, the Netherlands. This plant primarily treated domestic wastewater. Nitrifying activated sludge was cultivated in a glass-constructed reactor with feedback of biomass (Fig. 1). The reactor consisted of an aeration vessel (C) with a capacity of 0.36 l, from which the liquor was passed continuously to a settler (D) with a capacity of 0.08 l. The reactor was started by ®lling the system with activated sludge. Euent that left the apparatus was collected in a container (E). Aeration was achieved with an approximate air¯ow of 10 l hÿ1 through a capillary leading to the bottom of the aeration vessel. The nitrifying sludge was grown in a mineral salts medium: 4 g lÿ1 (NH4 )2 SO4 ; 0.2 g lÿ1 MgSO4 7H2 O, 1.5 g lÿ1 K2 HPO4 0.08 g lÿ1 CaCl2 and 0.1 ml lÿ1 of a
Fig. 1. Schematic diagram of the continuously fed activated sludge (CAS) reactor used for the laboratory-scale tests. The unit consists of a storage vessel (A), dosing pump (B), aeration section (C), settling section (D), collecting vessel (E), and air supply (F).
trace solution (Vishniac and Santer, 1957). The ¯ow of the mineral salts medium through the reactor was maintained by using a peristaltic pump at a ¯ow rate of 250 ml dÿ1 . Daily, 18 ml of sludge was removed from the aeration basin to maintain a sludge retention time of 20 days. The concentration of dissolved oxygen was between 2 and 7 mg lÿ1 . The pH in the unit was controlled at 7.5 by automatic titration with 20 g lÿ1 NaHCO3 . The sludge was incubated in diuse light at 20°C. 2.3. Analyses The EE2 content was analysed using HPLC. The HPLC consisted of a pump (Waters 510), a Novapak C18 (3:9 150 mm2 ) column and a UV detector (Separation 785A; LSC detector (bRAM)). The concentration of EE2 was measured with radiochromatography. The mobile phase was acetonitrile/water (40/60, v/v). The ¯ow rate was 1 ml minÿ1 . Samples (50 ll) were injected after sedimentation of the sludge. Ammonium was determined by forming a greencoloured indophenol with salicylate and hypochlorite in an alkaline medium. The absorbance of the complex formed was measured spectrophotometrically at 580 nm. The dry weight (DW) of the sludge was determined by ®ltering a 10 ml sample through a preweighed 12 lm cellulose nitrate membrane ®lter and drying this ®lter at 104°C to a constant weight. 2.4. Batch experiments Activated sludge was withdrawn from the continuously fed reactor or the full-scale reactor and injected into Erlenmeyer ¯asks. Approximately 0.1 mCi (3.7 MBq) [3 H]-ethinyl estradiol in 0.1 ml ethanol was added to the 15 ml sludge in an Erlenmeyer ¯ask. The initial cell density was 1.0 g lÿ1 and the initial EE2
J.S. Vader et al. / Chemosphere 41 (2000) 1239±1243
concentrations were approximately 50 lg ÿ1 l. To provide an exogenous source of energy hydrazine was added to the Erlenmeyer ¯asks at a concentration of 10 mg lÿ1 . Erlenmeyer ¯asks were incubated on a magnetic shaker at 100 rpm and aliquots of 1 ml were taken over time. Aliquots withdrawn were analysed by liquid scintillation counting and HPLC pro®ling. Erlenmeyers containing 0.2 g lÿ1 of nitrifying sludge were shaken at 100 rpm at 20°C for 4 h, to allow almost complete oxidation of the 50 mg ÿ1 l ammonium added. Samples from the Erlenmeyer ¯asks were centrifuged at 5.000 g for 5 min to remove sludge particles prior to making analyses of ammonium.
3. Results and discussion Nitrifying micro-organisms were enriched in a laboratory-scale activated sludge system fed with 4 g lÿ1 (NH4 )2 SO4 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 gÿ1 DW hÿ1 . A representative experiment of EE2 degradation by nitrifying sludge is shown in Fig. 2. This nitrifying sludge degraded EE2 completely within six days and did not require an adaptation period. The initial speci®c EE2 degradation rate by nitrifying sludge was 1 lg gÿ1 DW hÿ1 . Nitrifying activated sludge retained its maximum EE2 degradation abilities for approximately two days. After this period the rate of degradation levelled o, probably due to the anity of the micro-organisms for EE2 at low concentrations in the experiment or to a decrease in the activity of the non-growing cells (Fig. 2). The EE2 biodegradation was con®rmed in two similar experiments. No detectable degradation of EE2 present at a concentration of 50 lg lÿ1 was measured with activated sludge capable
Fig. 2. Degradation of EE2 by nitrifying sludge.
1241
of oxidising ammonium at a rate of only 1 mg NH4 gÿ1 DW hÿ1 . Nitrifying sludge was capable of degrading EE2 in the absence of ammonium. Nitrifying bacteria like Nitrosomonas use ammonium as an energy source and oxidise ammonium to nitrite. As oxidation of ammonium by a monooxygenase requires a source of reducing energy (NADH), the capacity to transform EE2 by the sludge may become a function of the available reducing energy. When hydrazine (10 mg lÿ1 ) was added as an external electron donor for NADH generation, it removed the reducing energy limitation. Nitrifying sludge supplied with hydrazine exhibited slightly higher EE2 degradation rates than sludge without hydrazine. This result indicates that EE2 degradation is mediated by ammonium monooxygenase activity. Ammonium-oxidising bacteria are able to co-metabolise many low molecular weight organic compounds (Hyman and Wood, 1983; Hyman et al., 1988; Rashe et al., 1990a,b; 1991; Juliette et al., 1993; Hyman et al., 1994; Keener and Arp, 1994). Our results demonstrate that nitrifying bacteria not only acts on low molecular weight organic compounds but also on a synthetic steroid. Although the removal of the parent compound already demonstrates the biological degradation of EE2 . the oxidation of EE2 was con®rmed by the formation of hydrophilic organic compounds, as can be seen from early eluting peaks in the radioactivity pro®le (Fig. 3). The pathway of ammonium oxidation is initiated by the enzyme ammonium monooxygenase. Monooxygenases can carry out reactions which insert oxygen into C±H bonds. Ammonium monooxygenase catalyses, for instance, the hydroxylation of alkanes to produce primary and secondary alcohols (Hyman and Wood, 1983; Hyman et al., 1988). The activity of nitrifying activated sludge probably results in hydroxylation, converting EE2 into hydrophilic products. A portion of the very polar radioactivity detected in the radio HPLC was quite volatile (probably tritiated water), indicating extensive hydroxylation of EE2 by the nitrifying activated sludge. These hydroxylated degradation products are essentially devoid of estrogenic activity (Bergink et al., 1983). Nitrogen removal in biological treatment systems is achieved by nitri®cation of all nitrogen to nitrate followed by removal of nitrate through denitri®cation. At present limited data of monitoring studies are available relating the nitrifying activity to the removal of EE2 in wastewater treatment plants. The importance of such data is illustrated by monitoring studies performed by Desbrow et al. (1998) and Belfroid et al. (1999) showing remarkable seasonal changes with respect to EE2 removal. In summer, the content of EE2 in the euents was below the detection limit of 0.2 ng lÿ1 in all four plants situated in England (only one sample of one plant contained an exceptionally high EE2 concentration). In
1242
J.S. Vader et al. / Chemosphere 41 (2000) 1239±1243
Fig. 3. HPLC chromatograms showing the degradation of EE2 and formation of hydrophilic products by nitrifying sludge. HPLC pro®les were from samples taken at the start (above) and the end (below) of a batch experiment. EE2 elutes at 8.5 min.
winter, EE2 concentrations detected ranged from 0.2 to 4.3 ng lÿ1 in two plants. The euent of a third plant monitored in winter contained no EE2 . Comparable seasonal changes were found in treatment plants in the Netherlands (Belfroid et al., 1999). Reduced nitri®cation in biological treatment systems at low temperatures due to wash-out of nitrifying bacteria is a well-known phenomenon. An adequate sludge retention time in relation to the growth rate ensures that the nitrifying bacteria do not wash out, which means that nitri®cation will take place. The higher growth rate of nitrifying bacteria in summer due to increased temperatures enables nitrifying bacteria to maintain themselves at lower sludge retention times. The results of the monitoring studies indicate that the ability to degrade EE2 is correlated with a temperature-dependent microbial processes such as nitri®cation. Although partial removal of ammonium during the winter period in the plant in the Netherlands (Belfroid, personal communication) seems to contradict this correlation, future monitoring studies of waste water treatment plants should record data on the capacity to nitrify and the sludge retention time. Nitrifying bacteria not only occur in biological wastewater treatment systems but also abundantly in natural ecosystems. The process of nitri®cation is considered to be a crucial process in the nitrogen cycle in natural ecosystems such as soils. EE2 degradation by nitrifying micro-organisms is a cometabolic process.
Cometabolic conversions are thought to play a signi®cant role in the degradation of xenobiotic compounds in nature (Alexander, 1994). In summary, nitrifying bacteria are widespread in the environment. As they can degrade EE2 without prior adaptation, it can be concluded that these bacteria represent a sink for EE2 in various environmental compartments and activated sludge systems. Acknowledgements We thank J.P. Sumpter for helpful comments. References Alexander, M., 1994. Biodegradation and bioremediation. Academic Press, San Diego, USA, pp. 177±195. Belfroid, A.C., van der Horst, A., Vethaak, A.D., Sch afer, A.J., Rijs, G.B.J., Wegener, J., Co®no, W.P., 1999. Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in the Netherlands. Sci. Total Environ. 225, 101±108. Bergink, E.W., Kloosterboer, H.J., van der Velden, W.H.M., van der Vlies, J., Winter, M.S., 1983. Speci®city of an estrogen binding protein in the human vagina compared with that of estrogen receptors in dierent tissues from dierent species. In: Jasonni, V.M. (Ed.), Steroids and
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