Determination of Estrogens in Sludge and Sediments by Liquid

Anal. Chem. 2002, 74, 3498-3504

Determination of Estrogens in Sludge and Sediments by Liquid Extraction and GC/MS/MS Thomas A. Ternes,*,† Henrik Andersen,‡ Daniel Gilberg,§ and Matthias Bonerz†

ESWE-Institute for Water Research and Water Technology, D-65201 Wiesbaden, Soehnleinstrasse 158, Germany, The Royal Danish School of Pharmacy, Institute of Analytical and Pharmaceutical Chemistry, Section for Environmental Chemistry, Universitetsparken 2, DK-2100 Copenhagen, Denmark, and Ecotoxicology GmbH, Bo¨ttgerstrasse 2-14, D-60437 Flo¨rsheim am Main, Germany

Two methods have been developed that enable the determination of estrogens down to 2 ng/g in digested and activated sludge from domestic sewage treatment plants (STPs) and down to 0.2 ng/g in freshwater sediments. The method for sludge analysis consists of solvent extraction; a gel permeation chromatography (GPC) cleanup step, a 1 g silica gel column; and finally, detection by GCion trap MS/MS of the silylated estrogens with MSTFA. For sediments, the solvent extraction was successively followed by silica gel cleanup, solid phase enrichment (SPE), and a HPLC cleanup before derivatization and GC/ MS/MS detection. Mean recoveries of the estrogens mainly exceeded 70% in sludge and 90% in sediments. In activated and digested sewage sludge, estrone and 17βestradiol were detected up to 37 ng/g and 49 ng/g, respectively, and 17r-ethinylestradiol up to 17 ng/g. The occurrence of estrogens in digested sludge indicates that estrogens can be persistent during sludge digestion. In river sediments, estrone and 17β-estradiol were detected up to 2 ng/g (estrone), and the contraceptive 17rethinylestradiol was found with a maximum of 0.9 ng/g. Mestranol, a prodrug for 17r-ethinylestradiol, was not detected either in sludge or in sediments. Endocrine-disrupting effects in the aquatic environment, such as the feminization of male fish can probably be attributed to the presence of estrogens in river water.1-3 Many other substances, such as alkylphenol, phthalic esters, PCBs, and phytoestrogens are suspected to influence the hormonal system, as well.4 Estrogens are extremely potent compounds and estrogenic effects have been observed in laboratory studies down to 1 ng/L.3 Worldwide, in municipal sewage treatment plant (STP) discharges * Corresponding author. Phone: 49 611 7804343. Fax: 49 611 7804375. E-mail: [email protected]. † ESWE. ‡ The Royal Danish School of Pharmacy. § Ecotoxicology GmbH. (1) Sumpter, J. P.; Jobling, S. Environ. Health Perspect. 1995, 103, 173-78. (2) Desbrow, C.; Routledge, E. J.; Brighty, G. C.; Sumpter, J. P.; Waldock, M. Environ. Sci. Technol. 1998, 32, 1549-1558. (3) Routledge, E. J.; Sheahan, D.; Desbrow, C.; Brighty, G. C.; Waldock, M.; Sumpter, J. P. Environ. Sci. Technol. 1998, 32, 1559-1565. (4) Roembke, J.; Knacker, T.; Stahlschmidt-Allner, P. Study about enviromental problems in context with drugs. F+E Vorhabens Nr. 106 04 121, Umweltbundesamt, Berlin, 1996.

3498 Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

and in the receiving waters, predominantly estrone, 17β-estradiol, and the contraceptive 17R-ethinylestradiol have been detected. Concentration levels of only a few nanograms per liter or even down to the picograms-per-liter range2,5-11 have been reported. The log KOW of 3.1-4.7 indicates that estrogens are rather lipophilic and should appreciably adsorb onto sediment and sludge (Table 1). This assumption is underscored by the detection of high concentrations of estrogens in water released by dewatering sewage sludge.12 Therefore, a potential contamination of soil with estrogens may be caused by the application of digested sludge from municipal STPs onto agricultural fields. Further, it seems likely that estrogens are present in sediments, and because of their extremely high estrogenic potency, the possible threat to sediment biota cannot be ruled out. Analytical methods for the determination of estrogens have been described in the literature for aqueous matrixes, such as wastewater and river water. The methods published for the determination of estrogens in water are frequently based on SPE, silylation and detection by GC/MS or GC-ion trap MS/MS. The LOQs are in the lower nanograms-per-liter range for such methods. The authors used different agents for silylation, such as MSTFA,13 hexamethyldisilazane/trimethylchlorosilane/pyridin, BSTFA, and MSTFA/TMCS.14,15 Acetylation with anhydrides, (e.g. heptafluorobutyric anhydride) is the other frequently applied derivatization technique.16 Kuch and Ballschmiter17 recently (5) Baronti, C.; Curini, R.; D’Ascenzo, G.; Di Corcia, A.; Gentili, A.; Samperi, R.; Environ. Sci. Technol. 2000, 34, 5059-5066. (6) Ternes, T. A.; Stumpf, M.; Mueller, J.; Haberer, K.; Wilken, R.-D.; Servos, M. Sci. Total Environ. 1999, 225, 81-90. (7) Spengler, P.; Metzger, J. W.; Ko ¨rner, W. Environ. Toxicol. Chem. 2001, 20, 2133-2141. (8) Belfroid, A. C.; Van-der, Horst A.; Vethaak, A. D.; Schafer, A. J.; Rijs, G. B.; Wegener, J.; Cofino, W. P. Sci. Total Environ. 1999, 225, 101-08. (9) Johnson, A. C.; Belfroid, A.; Di Corcia, A. Sci. Total Environ. 2000, 256, 163-173. (10) Huang, C. H.; Sedlak, D. L. Environ. Toxicol. Chem. 2001, 20, 133-139. (11) Snyder, S. S.; Keith, T. L.; Verbruegge, D. A.; Snyder, E.; Gross, T. S.; Kannan, K.; Giesy, J. P. Environ. Sci. Technol. 1999, 33, 2814-2820. (12) Matsui, S.; Takigami, H.; Matsuda, T.; Taniguchi, N.; Adachi, J.; Kawami, H.; Shimizu, Y. Water Sci. Technol. 2000, 42, 173-179. (13) Sturm, G.; Loof, I.; Spa¨tling, L.; Mohr, K.; Buchholz, R. Anal. Chem. Symp. Ser. 1981, 10, 315-318. (14) Fukushima, S.; Akane, A.; Matsubara, K.; Shiono, H.; Morishita, H.; Nakada, F. J. Chromatogr. 1991, 565, 35-44. (15) Daeseleire, E.; Vanoosthuyze, K.; Van Peteghem, C. J. Chromatogr. A. 1991, 674, 247-253. (16) Lee, H. B.; Pert, T. E. J. - Assoc. Off. Anal. Chem. Int. 1999, 81, 12091216. 10.1021/ac015717z CCC: $22.00

© 2002 American Chemical Society Published on Web 05/24/2002

Table 1. Chemical Structures, CAS Numbers and log Kow

reported another interesting derivatization procedure. They converted the estrogens to pentafluorobenzoylate esters, which were detected by GC/NCI-MS. The derivatization step can be avoided by using liquid chromatography,18,19 whereby comparable detection limits to GC/ion trap MS can be attained using LC tandem MS.5 However, even LC single MS can lead to sufficient detection limits when a very specific cleanup step is applied, such as the separation of the natural estrogens by monoclonal antibodies.20 However, no methods are published that describe the determination of estrogens in sludge and river sediments down to the lower nanograms-per-gram range. The aim of the current study was to develop reliable methods to determine natural estrogens and two contraceptives in digested and activated sludge as well as in river sediments. EXPERIMENTAL SECTION Sampling of Sewage Sludge and Sediments. The activated sludge was obtained by filtration of the slurry of activated sludge samples randomly taken from German STPs. The digested sludge was taken from the respective digester of the STPs. The following is a description of the STPs where the sludge samples were taken. STP-I: preliminary clarification, aeration tank; denitrification; phosphate removal by addition of Fe(II)Cl2; final clarification; population equivalent, 350 000. STP-II: preliminary clarification, aeration tank; final clarification; population equivalent, 400 000. The sediment samples were randomly taken from the sediment surface of the stream by a gripping device (van Veen) which in general takes a sample at depths up to 5 cm. Extraction of Sludge and Sediments. Sludge and sediments were freeze-dried, and a sample (0.5 g sludge or 5 g sediment) was taken and extracted with 4 mL and 3 mL of methanol and subsequently two times with 3 mL of acetone. For each extraction step, the slurry of the sample was ultrasonicated for 10 min. The slurry was then centrifuged at 3000 rpm for 5 min, and the (17) Kuch, H. M.; Ballschmiter, K. Environ. Sci. Technol. 2001, 35, 3201-3206. (18) Cannell, G. R.; Mortimer, R. H.; Maguire, D. J.; Addison, R. S. J. Chromatogr. 1991, 563, 341-347. (19) De Alda, M. J. L.; Barcelo, D. J. Chromatogr. 2000, 892, 391-406. (20) Ferguson, P. L.; Iden, C. R.; McElroy, A. E.; Brownawell, B. J. Anal. Chem. 2001, 73, 3890-3895.

supernatants were collected. Eventually, the four solvent fractions were combined and then evaporated in a fume hood to dryness by a gentle nitrogen stream. The surrogate standard, 17β-estradiol17β-acetate from Sigma (Deisenhofen, Germany), was spiked into the slurry of the first extracted fraction with 4 mL of methanol. Preparative Gel Permeation Chromatography (GPC) for Sludge. Matrix components with high molecular masses (∼>1000 amu) were removed by GPC. The sludge extracts were dissolved in 5 mL acetone/cyclohexane (1:3, v/v) and filtered through a 25 mm × 0.45 µm PTFE filter (Supelco, Bellefonte, PA), and 4.5 of the 5.0 mL was injected onto the GPC column. The GPC system used was an abc Autoprep 1000 with an abc GPC/Sample unit (abc Instruments, Columbia, MO) equipped with a special GPC PTFE column (GPC Prep Column Serial 000117-601-OP) from O‚I‚Analytical (College Stadium, TX) filled with Bio-Beads SX-3. A mixture of acetone/cyclohexane (1:3, v/v) was used as solvent, and the flow rate was adjusted to 4.0 mL/ min. The first fraction, from 0 to 14 min, which contains highermolecular-weight compounds, was discarded. The second fraction containing the estrogens, was collected between 14 and 27 min. Afterward, the GPC column was rinsed for 5 min with the eluent prior to the next injection. The volume of the collected fraction was reduced to ∼1 mL with a rotary evaporator at 20 kPa and 40 °C. Finally, the sample extracts were evaporated to dryness by a gentle N2 stream and dissolved in 200 µL of hexane/acetone (65: 35, v/v). The rotary evaporator has to be placed behind a protecting shield to protect the operator from a potential implosion that may occur due to the vacuum. Silica Gel Cleanup for Sludge and Sediment. After heating for 8 h at 150 °C, the silica gel (silica gel 60, 70-230 mesh, from Merck, Darmstadt, Germany) was deactivated with 1.5% water (w/w). A 1-g portion of silica gel was stirred in 5 mL of hexane/ acetone (65:35, v/v). The slurry was manually filled into a 6 mL glass cartridge. The sludge extracts (after GPC cleanup) and the sediment extracts (after solvent extraction) were quantitatively transferred to the prepared 1 g silica gel column. The analytes were eluted using 5 mL of hexane/acetone (65:35, v/v), which was then evaporated in a fume hood to a volume of ∼300 µL by a gentle nitrogen stream. Solid-Phase Extraction of Sediment Extracts. The solidphase material, 500 mg of RP-C18 (Separtis GmbH, GrenzahlWyhlen, Germany), was filled into glass cartridges and then conditioned with 3 × 2 mL hexane, followed by 1 × 2 mL acetone, 3 × 2 mL methanol, and 5 × 2 mL water (pH 7). The sediment extract of the silica cleanup ( ∼300 µL) was mixed with 100 mL of groundwater that had 1.3 g/L of tetrabutylammoniumbromide added. The groundwater used is not influenced by anthropogenic contamination. The samples were passed without filtration through the packed glass cartridges at a flow rate of ∼20 mL/min. Subsequently, the solid phase was dried completely by a nitrogen stream for 1 h, and the analytes were eluted four times with 1 mL of acetone. The acetone extracts were evaporated in a fume hood to 200 µL by a gentle nitrogen stream. HPLC Cleanup of Sediment Extracts. The semipreparative HPLC system consisted of a Bishop HPLC compact pump 2250; a Bischoff UV/vis Lambda 1010 UV detector; and a fraction collector, Gilson FC 203 B. HPLC conditions were as follows: the column was a 250 × 8 mm Kromasil 100 C18 (5 µm) with a Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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Table 2. Precursor, Product Ions, and Retention Times Used in GC/MS/MS Detection substances

retention time, min

precursor ion, m/z

product ion 1, m/z

product ion 2, m/z

CID resonant amplitude, V

mirex estrone 17β-estradiol mestranol 17β-estradiol-17β-acetate 17R-ethinylestradiol

21.14 22.19 22.80 23.93 24.44 25.28

272 342 416 367 386 425

257 326 349 326 231

244 285 193 297 193

0.31 0.37 0.37 0.37 0.31

precolumn 40 × 8 mm Kromasil 100 C18 (5 µm), both purchased from A-Z Analytical, Langen, Germany. An isocratic flow of 2 mL/min of a water/acetonitrile eluent (20:80, v/v) was used at room temperature. The acetone extract of SPE was filled up to ∼350 µL with acetonitrile/water and ∼230 µL was injected onto the HPLC column. The first fraction was collected between 4.5 and 6.7 min (estrone, 17β-estradiol, mestranol); the second, between 9.3 and 10.6 min (17β-estradiol-17β-acetate); and the last fraction, between 11 and 12.5 min (17R-ethinylestradiol). The fractions were combined, and the volume was reduced to ∼1 mL with a rotary evaporator at 40 °C and 10 kPa. Afterward, the sample extracts were evaporated to dryness by a gentle nitrogen stream and derivatized as described below. Skin contact to acetonitrile should be avoided in this procedure by using gloves. Furthermore, the HPLC equipment should be placed in a fume hood or at least a suction point should be placed close to the fraction collector to minimize acetonitrile fumes released into the surrounding laboratory. Derivatization. For the detection by GC/MS/MS,6 the sample extracts were derivatized by adding 50 µL of the derivatization mixture (N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA)/ trimethylsilylimidazole (TMSI)/dithioerytrol (DTE), (1000:2:2; v/v/w). MSTFA and TMSI were purchased from Sigma (Deisenhofen, Germany) and DTE from Merck (Darmstadt, Germany). After a reaction time of 1 h at 60 °C, the solution was evaporated to dryness by a gentle nitrogen stream, and the residue was dissolved in 200 µL of hexane. Finally, as an instrumental standard, 100 ng of mirex (from Promochem, Wesel, Germany) was added to the final extract. The silylation mixture has to be handled in a fume hood, and skin exposure has to be strongly avoided. GC and MS/MS Detection-Operating Conditions. Separation and detection of the analytes was achieved using a GC/MS/ MS system, a Varian GC 3400 coupled to a Varian Saturn 4D mass spectrometer. The gas chromatograph was equipped with a PTV injector, which was coupled to a retention gap (uncoated, deactivated, 1 m × 0.32 mm, Hewlett-Packard) and a capillary column (Restek, Bad Soden, Germany; XTI-5/30 m × 0.25 mm × 0.25 µm). The head pressure was 60 kPa He. The retention times, the selected ion masses, and the collision induced dissociation (CID) resonant amplitudes are listed in Table 2. GC injection parameters: 5 µL, splitless; 50 °C; 100 °C/min to 300 °C, 300 °C isothermal 10 min. GC temperatures: 50 °C isothermal for 3.5 min, 20 °C/min to 240 °C, 2 °C/min to 290 °C, and 290 °C isothermal for 10 min. MS parameters: temperature of transfer line (direct interface), 280 °C; ion source, EI mode; electron energy, 70 eV; ion trap 3500

Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

temperature, 250 °C; collision-induced dissociation (CID) RF, 120 V; and window for MS/MS, 3 m/z. Determination of Recoveries. Freeze-dried sludge and sediments were spiked with the natural estrogens 17β-estradiol, estrone, and the contraceptives 17R-ethinylestradiol and mestranol (all were purchased from Sigma, Deisenhofen, Germany), which were dissolved in a stock solution (each with 10 µg/mL in methanol). After spiking, the samples were stirred intensively to spread over the spiking solution in the sample and to enable a sufficient contact of the estrogens with the matrix. For sediments, an additional ∼2 mL of acetone was added after spiking; otherwise, an equal distribution of the spiked compounds with the matrix would be difficult to achieve. The spiked samples were placed in a fume hood overnight at room temperature. The solvent extraction took place the next day (14 h later) to ensure that the acetone/methanol were completely evaporated and there was enough time at room temperature for sorption of the estrogens to sludge and to sediment constituents. The recoveries were determined in relation to a nonenriched standard solution, which was only derivatized. For calculation of the recoveries, mirex (nonenriched and nonderivatized instrumental standard) was used, which was spiked in the final volume. To evaluate directly the losses caused by the solvent extraction, 14C-labeled 17R-ethinylestradiol (labeled at C-1,2 in the phenolic ring; specific radioactivity, 5.54 MBq/g), a courtesy of Schering, Germany, was spiked to sludge and sediments, and extracted the next day ( ∼14 h later). The total radioactivity in the solvent extract was measured by a scintillation counter (LSC-Counters: Tricarb 2500 TR, Packard, Dreieich, Germany). Please note that experiments with 14C-labeled compounds should only be performed in special radioactivity laboratories. For the other cleanup steps, the individual recoveries were determined without matrixes by spiking the respective solvent with the estrogens. Solvent extraction, GPC, silica gel cleanup, SPE, HPLC-cleanup, derivatization, and detection by GC/MS/MS were performed as described above. Calibration, Detection Limits, and Blank Samples. The calibration was performed over the whole procedure after spiking the extraction solvent mixture with the reference standards. For sludge, an eight-point calibration was used ranging from 1 ng/g to 200 ng/g. For sediments, the calibration ranged from 0.1 to 200 ng/g, with 10 calibration points. Limits of quantification (LOQ), allowing for the quantification of analytes in native samples, were set as the second lowest calibration point within the linear correlation curve. The signal/noise ratio of the samples extracts has to be higher than 10. As blank samples, a methanol/ acetone mixture spiked with the surrogate standard 17β-estradiol17-acetate was included in each series of the analysis. The

Figure 1. Schemes of the analytical methods for estrogens in sludge of municipal STPs and in river sediments. Table 3. Recoveries of Various Estrogens in Sludge and Sediment with 95% Confidence Intervals after Subtracting the Original Contaminationa recovery, % digested sludge

estrone 17β-estradiol mestranol 17R-ethinylestradiol

activated sludge

sediment

absoluteb

relativec

absoluteb

relativec

absoluteb

recovery

recovery

recovery

recovery

recovery

relativec recovery

77 ( 11 66 ( 10 71 ( 8 57 ( 15

117 ( 10 117 ( 16 121 ( 17 94 ( 19

104 ( 11 73 ( 18 99 ( 12 99 ( 25

119 ( 3 83 ( 13 113 ( 5 113 ( 19

94 ( 37 97 ( 11 99 ( 26 99 ( 23

118 ( 34 110 ( 15 98 ( 513 105 ( 28

a Sludge spiked at 100 ng/g, n ) 6; sediment spiked at 4 ng/g; n ) 4. b Calculated using the instrumental standard mirex spiked in the final volume. c Calculated using the surrogate standard 17β-estradiol-17β-acetate spiked at the beginning. 3 n ) 3 (t-value was 4.3 instead of 3.18).

estrogen concentration in native samples was calculated in relation to the surrogate standard, whereas the instrumental standard mirex was used to confirm the reliability of the GC/ion trap MS measurement. One crucial quality assurance point for the quantification of estrogens in real samples was the area ratio R of the surrogate standard and mirex (R ) Asurrogate/Amirex). A quantification of estrogens in native samples was only conducted if the ratio R of the sample extracts did not differ by more than 30% from the ratio R in the calibration. Thus, a significant loss during sample preparation could be mainly ruled out, since the instrumental standard mirex did not exhibit any losses, since it was spiked to the final extract. In the following, the term absolute recovery is addressed to values calculated with mirex, and the term relative recovery is addressed to values calculated with the surrogate standard 17βestradiol-17β-acetate. The latter compensated for losses during sample preparation, whereas the first exhibited the recovery over the whole method. Hence, mirex was used for recovery experiments and to confirm the quality of the GC/MS/MS detection,

and 17β-estradiol-17β-acetate was used for the quantification of native samples. All statistical errors are given as 95% confidence intervals. RESULTS AND DISCUSSION Recoveries and Detection Limits of the Analytical Methods. Sludge. The scheme of the analytical method for the analysis of estrogens in sludge is shown in Figure 1. Mean recoveries of the analytes in activated sludge ranged over the total method between 73 ( 18% and 104 ( 11% (Table 3) at a spiking level of 100 ng/g. In digested sludge, most of the analytes exhibited lower absolute recoveries with similar deviations; however, using the surrogate standard 17β-estradiol-17β-acetate, these losses were efficiently compensated, because the relative recoveries showed almost quantitative results for activated and digested sludge within 83 ( 13% and 121 ( 17%. Losses seem to be caused by the solvent extraction the recovery of which was ∼88 ( 17% for 17Rethinylestradiol after a contact time of 14 h, whereas GPC, silica gel, and SPE cleanup were nearly quantitative (Table 4). It has to Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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Table 4. Recoveries of Estrogens after Extraction with Methanol and Acetone of 0.5 g Sludge and 5.0 g Sediment Spiked with 15.5 ng/g and 4.0 ng/g 14C-labeled 17r-ethinylestradiol, Respectively, and Recoveries for Other Individual Cleanup Steps without Matrix Recovery % (95% Confidential Interval) sludge solvent sediment solvent extraction extraction (n ) 4) (n ) 4) first extraction (3 mL methanol) second extraction (2 mL methanol) third extraction (2 mL acetone) fourth extraction (2 mL acetone) sum

estrone 17β-estradiol mestranol 17R-ethinylestradiol

54.3 ( 8.3 22.2 ( 5.9 8.7 ( 2.5 3.2 ( 1.1 88 ( 17

73.1 ( 16.2 10.4 ( 2.4 1.7 ( 0.3 0.2 ( 0.3 85 ( 17

GPC (n ) 6)

Silica gel (n ) 4)

SPE (n ) 2)

HPLC (n ) 3)

100 ( 10 101 ( 8 98 ( 7 100 ( 11

109 ( 16 98 ( 10 109 ( 14 98 ( 12

97 ( 20 93 ( 17 99 ( 18 93 ( 6

98 ( 10 95 ( 10 98 ( 18 115 ( 32

be considered that for silica gel and GPC cleanup, the recoveries were determined without matrix. Therefore, losses in digested sludge might be explained by overloading or matrix interference on the columns. The extraction efficiency after 14 h could only be measured for 17R-ethinylestradiol, since the other estrogens were not available as 14C-labeled substances. The LOQs for the estrogens were 2 ng/g for estrone, 17β-estradiol, and 17Rethinylestradiol, and 4 ng/g for mestranol, considering a signal/ noise ratio of at least 10 and the second-lowest calibration point in the linear calibration curve. The linear calibration ranged from 1 to 200 ng/g, with a sufficient correlation coefficient (see Table 5). Sediment. The scheme of the analytical method for the analysis of estrogens in sediments is shown in Figure 1. Mean absolute recoveries of the estrogens at a spiking level of 4 ng/g always exceeded 90% (Table 3). The relative recoveries after compensation with the surrogate standard 17β-estradiol-17β-acetate also showed quantitative results within the statistical error. LOQs were down to 0.2 ng/g achieved for estrone and 17β-estradiol and down to 0.4 ng/g for the contraceptives. The individual recovery of the solvent extraction was 85 ( 17% for 14C-labeled 17R-ethinylestradiol. Silica gel and SPE cleanup were quantitative within the statistical uncertainty. Prior to the SPE enrichment, the extract of the silica gel cleanup was dissolved in 100 mL groundwater that had 1.3 g/L of tetrabutylammoniumbromide added. The change from the organic solvent to the water

is a very crucial step. Because of precipitation in the water phase, losses of ∼30% can occur as a result of sorption of the estrogens onto the flocs. Therefore, a filtration should be avoided. Without the final HPLC cleanup, the GC/MS/MS measurements of derivatized sample extracts were not reproducible. After just a few injections, the background increased significantly. Hence, the final HPLC-cleanup is an essential step for the developed method. Contamination of Native Sample. Sewage Sludge. In activated and digested sludge of two municipal STPs, the natural estrogens estrone and 17β-estradiol were detected up to 37 ng/g (estrone) and up to 49 ng/g (17β-estradiol). A GC/ion trap MS/MS chromatogram is shown in Figure 2. Because of the lack of a sufficient amount of data, a significant difference between the two STPs and between activated and digested sludge cannot be drawn. However, it can be concluded that natural estrogens were not totally eliminated in the digester. These results coincide with the relatively high estrogen equivalent detected with an E2-specific immunoassay and YES-estrogen assay by Matsui et al.12 in water released by dewatering sewage sludge. 17R-Ethinylestradiol was also extracted from activated and digested sludge with a concentration range between LOQ (n ) 4)

plant A

plant B

plant A

plant B

2 2 4 2

0.999 0.993 0.954 0.998

2 4 3 0