Environ. Sci. Technol. 2001, 35, 3201-3206
Determination of Endocrine-Disrupting Phenolic Compounds and Estrogens in Surface and Drinking Water by HRGC-(NCI)-MS in the Picogram per Liter Range HOLGER M. KUCH AND KARLHEINZ BALLSCHMITER* Department of Analytical and Environmental Chemistry, University of Ulm, Albert-Einstein-Allee 11, D-89069 Ulm, Germany
A method for the analysis of phenolic estrogenic active compounds in surface and drinking water in the picogram per liter range is described. Besides the widely used monomer bisphenol A, 4-tert-octylphenol [4-(1,1,3,3tetramethylbutyl)phenol] and the technical isomer mixture of 4-nonylphenol; phenolic steroid hormones such as the endogenous estrogens estrone, 17R-estradiol, and 17βestradiol; and the exogenous estrogen 17R-ethinylestradiol were determined in water at the 20-200 pg/L level. Water samples from 1 to 5 L were extracted by solidphase extraction (SPE) on a cartridge system containing LiChrolut EN as sorbent. The phenols and steroids were converted into their pentafluorobenzoylate esters in an extractive derivatization reaction. The derivatives were then determined by high-resolution gas chromatography with negative chemical ionization mass spectrometric detection (HRGC-(NCI)-MS) in the selected ion mode (SIM). All results were also confirmed by HRGC with electron capture detection (ECD). This highly sensitive and specific method gives a limit of detection (LOD) of 20 pg/L for bisphenol A and 4-tert-octylphenol in drinking water samples and 50 pg/L in STW effluent, respectively. The LODs for technical 4-nonylphenol, 17R-ethinylestradiol, and other estrogens are in the range of 50 pg/L in drinking water to 200 pg/L in STW effluent, respectively. In all river water samples in southern Germany, bisphenol A was found in concentrations ranging from 500 pg/L up to 16 ng/L, 4-nonylphenol was from 6 up to 135 ng/L, and the steroids were from 200 pg/L up to 5 ng/L. In drinking water, bisphenol A was found in concentrations ranging from 300 pg/L to 2 ng/L, 4-nonylphenol was from 2 to 15 ng/L, 4-tert-octylphenol was from 150 pg/L to 5 ng/L, and the steroids were from 100 pg/L to 2 ng/L. Mean recoveries over the whole analytical protocol, measured in bidistilled water, generally exceeded 70%. These results indicate that environmental endocrinedisrupting estrogens are not completely removed in the process of sewage treatment but are carried over into the general aquatic environment. After ground passage, they can eventually be found in drinking water.
Introduction Estrogenic effects of treated wastewater, spilling into the aquatic environment, were first verified by Purdom et al. in 10.1021/es010034m CCC: $20.00 Published on Web 06/28/2001
2001 American Chemical Society
1994 (1). In caged male rainbow trout, the mainly domestic effluents caused elevated levels of vitellogenin, a yolk protein that usually only female fish are able to produce (2). “Feminization” of male species due to environmental pollution has been a controversial issue since then. Sewage treatment works (STWs) permanently receive a complex mixture of industrial, domestic, and agricultural wastewater containing a load of synthetic and natural chemical compounds. It has been demonstrated that, because of incomplete removal or forming of an active form during the process of sewage treatment, endocrine-active chemicals are released in surface water like rivers, lakes, and seas or adsorbed to sewage sludge or sediment. Ternes et al. (3) were able to show in aerobic batch experiments that steroid conjugates such as glucuronides of 17β-estradiol are rapidly cleaved in contact with activated sludge, and thus the active form of the estrogen is released. To date, estrogenic effects on aquatic wildlife have not been conclusively linked to only one particular compound, but some chemicals are mainly made responsible for causing endocrine disruption. Among them, the natural estrogens estrone (E1), 17R-estradiol (aE2), and 17β-estradiol (bE2) and the exogenous 17R-ethinylestradiol (EE2), the active ingredient in oral contraceptive pills, possess the highest estrogenicity. Apart from these steroids, alkylphenols such as 4-tert-octylphenol [OP, 4-(1,1,3,3-tetramethylbutyl)phenol] and the technical isomer mixture of 4-nonylphenols (NP), both breakdown products of nonionic surfactants (4), and bisphenol A (BPA), a widely used monomer for epoxy resins and polycarbons, show estrogenic potentials of 4 and more orders of magnitude lower than bE2 (5). Therefore, they are considered as xenoestrogens. But also representatives of the groups of PCBs, dioxins, phytoestrogens, pesticides, preservatives, antioxidants, or phthalic esters contribute to the daily exogenous burden of humans and wildlife with hormonally active agents (6-9). Besides methods using radioimmunoassay (10, 11) for sensitive determination of the polar, phenolic compounds, a method of choice is HRGC-MS with electron impact ionization (EI) and selected ion monitoring (SIM) without derivatization (22) or after derivatization to trimethylsilyl ethers (12-16), methyl ethers (17, 18), acetyl esters (19), and pentafluorobenzoyl and heptafluorobutyl esters (20, 21). These methods commonly give a LOD of several nanograms per liter or slightly below. The predictable concentrations in surface and drinking water are expected in the lower nanogram per liter range for the phenols and in the lower picogram per liter range for the steroids. To monitor such levels in drinking water and groundwater, a method was developed to quantitate natural and synthetic estrogens down to a LOD of 20 pg/L. We have chosen negative chemical ionization (CH4-NCI) to extend the sensitivity of the GC-MS approach. Monitoring at these levels may prove essential for the risk assessment on endocrine disruption regarding aquatic species and human health, respectively.
Experimental Section Selection of Sampling Sites. In this study, three municipal STW effluents in southern Germany [Ulm (n ) 6), Langenau (n ) 8), and Blaubeuren (n ) 2)] were analyzed stochastically in the mornings from June to October 2000. One of the effluents tested (STW of Ulm, discharging into the Danube River) receives domestic as well as industrial but only little agricultural input. Its total capacity is 350 000 population * Corresponding author telephone: +49-731-502-2751; fax: +49731-502-2763; e-mail:
[email protected]. VOL. 35, NO. 15, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Abbreviations Implemented for All Compounds abbrev
common name
full name
E1b aE2b bE2b EE2b n-NPd NPa OPa BPAc BPAd16a BNDe BPFBBa
estrone 17R-estradiol 17β-estradiol 17R-ethinylestradiol 4-n-nonylphenol technical nonylphenol 4-tert-octylphenol bisphenol A bisphenol A-d16 binaphthyldiol bispentafluorobenzylbenzene
1,3,5(10)-estratriene-3-ol-17-one 1,3,5(10)-estratriene-3,17β-diol 1,3,5(10)-estratriene-3,17β-diol 17R-ethinyl-1,3,5(10)-estratriene-3,17β-diol 4-n-nonylphenol technical 4-nonylphenol (mixture of branched isomers) 4-(1,1’,3,3′-tetramethylbutyl)phenol 2,2’-bis-(4-hydroxyphenyl)propane 2,2’-bis-(4-hydroxyphenyl)propane-d16 (R)-(+)-1,1′-binaphthyl-2,2′-diol 1,4-bis(pentafluorobenzyl)benzene
a Aldrich (Steinheim, Germany); purum. b Sigma (St. Louis, MO); min 99% purity. c Fluka (Buchs, Switzerland); purum. Germany); purum. e Merck (Darmstadt, Germany); for synthesis.
equivalents with 200 000 inhabitants in the catchment area. The two other effluents (STW of Blaubeuren, discharging into the Blau River, and STW of Langenau, discharging into the Nau River) have a similar size of 15 000 population equivalents each and receive primarily domestic but also agricultural input. All the STWs use the common three-step treatment of wastewater consisting of preliminary clarification, an activated sludge step, and final clarification. River and creek samples of the Danube (n ) 13), Nau (n ) 4), and Blau (n ) 4) were taken simultaneously with the wastewater samples upstream and approximately 1 km downstream of the STW effluent sites. The Iller River (n ) 4), which mainly receives its water from the Alps, was sampled without a respective STW for comparison. Three creeks [Schussen, Laiblach, and Argen (n ) 2 each)] that flow into the Lake Constance, the largest drinking water reservoir for southern Germany, were also analyzed. Drinking water samples from three different sites in southern Germany were also investigated. Tap water samples were taken stemming from groundwater supplies of the Danube and surface water supplies of Lake Constance, respectively. Generally 1- or 2-L samples were taken out of STW effluents, while 2- or 5-L river and drinking water samples were investigated. All samples were collected in purified and annealed glass bottles with Teflon caps. The water samples were stored at 4 °C and extracted within at least 2 days; dried SPE cartridges were stored at 4 °C when they could not be eluted directly after extraction. Solid-Phase Extraction. In Table 1, all investigated compounds are listed including the abbreviations implemented in this work. All solvents and reagents used were of nanograde or picograde purity. A total of 100 mg of the ethinylbenzene-divinylbenzene copolymer LiChrolut EN (Merck, Darmstadt, Germany) per liter of water sample (at least 200 mg) was filled into specially designed glass cartridges (10 mm i.d.) with a 500-mL reservoir and connection to a vacuum line. The solid phase was then conditioned with 3 mL of acetone (picograde, Promochem, Wesel, Germany), followed by the same amount of methanol (picograde, Promochem, Wesel, Germany) and bidistilled water (pH 4). Water samples were adjusted to pH 4 by the addition of 300 µL of concentrated H2SO4 (p.a., Merck, Darmstadt, Germany) per liter and filtered through annealed glass wool when poured into the cartridges. Thirty nanograms of the recovery standards 4-n-nonylphenol (n-NP) and (R)-(+)-1,1′-binaphthyl-2,2′-diol (BND) in 100 µL of methanol each were added into the cartrigde, and the water samples were extracted at a flow rate of 10 mL/min. After being dried in a stream of air for 10 min, the analytes were eluted from the adsorbent with 5 mL of acetone followed by 5 mL of methanol. The solvents were then evaporated to 200 µL of methanolic phase in a rotavapor. 3202
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d
Riedel-de Haen (Seelze,
Derivatization for GC. Extractive derivatization was carried out by modifying a method used by Renberg et al. and Wahlberg et al. for the pentafluorobenzoylation of phenolic compounds in water (20, 21). Recently Nakamura et al. adapted a similar method for the HRGC-(NCI)-MS determination of alkylphenols in water (27). The eluate of SPE is transferred into a 10-mL reaction flask. A total of 30 ng of bisphenol A-d16 (BPA-d16) in 100 µL of methanol was added as a recovery standard in order to control the completeness of derivatization and the transfer into the organic phase. The solution is then evaporated to dryness with a rotary evaporator, and the residue is dissolved in 2 mL of bidistilled water. Then 50 µL of 2 M KOH and 10 µL of a 10% solution of pentafluorobenzoyl chloride (PFBCl) (for synthesis, Merck, Darmstadt, Germany) in toluene (nanograde, Promochem, Wesel, Germany) were added. The derivates were generated and extracted twice with 2 mL of hexane (unisolv, Merck, Darmstadt, Germany) by shaking manually for 2 min. The combined organic phases were finally spiked with 20 ng of 1,4-bispentafluorobenzylbenzene (BPFBB) in 100 µL of isooctane (picograde, Promochem, Wesel, Germany) as an internal standard for quantification and evaporated to 100 µL. To control the derivatization, PFBCl was added to the remaining aqueous phase and extracted with 2 mL of hexane. No signals in GC-MS analysis could be detected. Thus, the derivatization procedure was presumed to be about quantitative. This extractive derivatization also provides a very effective matrix separation step. Polar humic substances remain in the yellow colored aqueous phase and yield a colorless sample for GC analysis. Determination by HRGC-(CH4-NCI)-MS (SIM). Gas chromatography and detection were performed on a HP 6890 series gas chromatograph with ALS on-column injection coupled to a HP 5973 MSD (Hewlett-Packard, Wilmington, DE) equipped with CI source. Data acquisition was carried out by aid of a HP Kayak Chemstation. Chromatographic Conditions. DB5MS (J&W Scientific, Folsom, CA) 60 m × 0,32 mm × 0,25 µm + 2 m retention gap; He 2 mL/min constant flow mode; injection volume 2 µL cold on-column; (80 °C, 3 min), 10 °C/min (200 °C, 1 min), 30 °C/min (245 °C, 10 min), 0,45 °C/min (260 °C, 1 min), 30 °C/min (300 °C, 10 min). MSD Parameters. NCI mode; reagent gas methane, flow 2 mL/min; transfer line temperature 310 °C; ion source temperature 150 °C; quadrupole temperature 150 °C; for SIM masses, see Table 2. Determination by HRGC-ECD. All samples were also analyzed by HRGC with electron capture detection (ECD). A HP 6890 series gas chromatograph equipped with ALS oncolumn injection and µ-ECD (Hewlett-Packard, Wilmington, DE) was used. Data acquisition was carried out by a HP Vectra Chemstation.
TABLE 2. Masses (m/z) of PFB Derivatives Used for HRGC-(NCI)-MS (SIM) Quantification (QM), Supplementary Identifier Masses (IM), and Characteristic Fragments in EI NCI
EI
MW QM I QM II IM I IM II E1 aE2 bE2 EE2 n-NP NP OP BPA BPAd16 BND IS BPFBB
464 466 466 490 414 414 400 616 630 674 466
464 466 466 490 414 414 400 616 630 674 466
465 467 467 491 415 415 401 617 631 675 467
I
II
III
IV
448 195 407 420 464 448 195 407 420 466 448 195 407 420 466 474 472 195 407 354 490 396 195 301 414 396 195 315 329 343 382 195 329 400 406 195 601 602 616 420 195 612 613 630 656 479 195 674 675 452 448 195 299 466
Chromatographic Conditions. RTX 1701 (Restek, Bad Soden, Germany) 30 m × 0,32 mm × 0,25 µm + 2 m retention gap; H2 2 mL/min constant flow mode; injection volume 2 µL cold on-column, ECD temperature 300 °C; (80 °C, 3 min), 10 °C/min (200 °C, 1 min), 30 °C/min (245 °C, 5 min), 0,5 °C/min (260 °C, 5 min) 30 °C/min (280 °C,10 min). Recoveries and Blank Samples. For the determination of recoveries, bidistilled water was spiked with a stock solution containing the individual steroids and phenols in methanol. The resulting concentrations were varied and ranged between 200 pg/L and 4 ng/L for BPA, OP, and NP and between 1 and 20 ng/L for the steroids. The 1-L blank samples of bidistilled or reverse osmosis water were only spiked with the recovery standards n-NP, BND, and BPA-d16 and analyzed. Calibration and Quantification. Stock solutions containing all analytes at acurately defined concentrations were made in methanol by dilution. Specific amounts of these stock solutions were derivatized as described above. Quantification was carried out by calculation of the relative response factors (RRF) based on the area of the quantification standard BPFBB. The RRFs were calculated by integrating the SIM tracks (see Table 2) of four stock solutions with concentrations of 1-4, 10-40, 100-400, and 1000-4000 pg/µL of the analytes. BPA and OP have the highest response in NCI-MS determination and were therefore added in lower concentrations (1, 10, 100, and 1000 pg/µL, respectively); aE2 and bE2 had the lowest response and thus highest concentrations (4, 40, 400, and 4000 pg/µL, respectively). The isomer mixture of NP was quantified by integrating the SIM tracks of four selected, characteristic signals that were present in all standard solutions and samples. Signals for the limits of detection (LODs) and the limits of quantification (LOQs) were set as the 3- and 6-fold height of noise, respectively.
Results Recoveries and Blanks of the Analytical Protocol. Mean recoveries of all analytes in bidistilled water generally exceeded 70% at all spiking levels. Recoveries of the steroids lay in the range between 71% and 79%, with exception of the estradiols (between 56% and 67%). Recoveries for the phenols were found to be in the range of 89-92%, with the exception of BPA and BND (70-79%). The relative standard deviation (RSD) varied from 9% to 15% and indicated a satisfactory reproducibility and precision of the whole analytical protocol. Surprisingly, OP, NP, and BPA could be detected even in 1-L blank samples of bidistilled or reverse osmosis water, although at levels (200-400 pg/L) where their occurrence had no significant influence on the determination of recoveries at the applied spiking levels. In the whole analytical process only annealed glass or steal tools and flasks (350 °C,
24 h) were used and flushed with acetone and methanol before use. Contamination by traces of dishwasher surfactants, a probable source for NP and OP, could be ruled out in this manner. All solvents and reagents were found to be free of all analytes by NCI-MS after derivatization. Thus blanks did not result from any chemical or material used. This is also demonstrated by the fact that blanks correlated well with the amount of purified water used for SPE. HRGC-(NCI)-MS Analysis. By introducing 5 (in the case of monophenols and estrogens) and 10 fluorine atoms (in the case of two phenolic groups), respectively, the analytes are turned into highly electrophilic derivatives. Electron capture and dissociative electron capture can be used as dominant ionization processes in opposition to trimethylsilylated compounds. Negative chemical ionization mass spectrometry (CH4NCI-MS) can therefore be used as well as electron capture detection (ECD) for very sensitive determination of absolute femtogram amounts. Another advantage of NCI over EI is its “soft” ionization, yielding a dominant molecular ion [M]*and the C13 isotope ion [M + 1]*- as the second intensive one. Thus, for quantitative analysis these two ions were used for all investigated compounds. Besides [M]*- and [M + 1]*-, other characteristic fragments occurred incidentally with significant abundance. They were used as supplementary identifier masses (see Table 2). In Figure 1, the EI and NCI mass spectra of BPA and EE2 as their PFB derivates are presented. In EI, the dominant fragment ion is always m/z 195, the pentafluorocarbonyl group (F5C7O*+), accompanied by several characteristic fragment ions of steroid and phenol ring fragmentation. HRGC-ECD Analysis. HRGC-ECD analysis of all samples was used for chromatographic identification on a second stationary phase (RTX 1701) and qualitative confirmation of the results obtained by HRGC-(CH4-NCI)-MS (SIM). As a result of the complex matrix and despite removal of humic substances during derivatization, with ECD detection a significant background noise and unidentified peaks still interfered with the analytes’ signals. Therefore, quantitative analysis was only performed by HRGC-(CH4-NCI)-MS. STW Effluents and River Water. Figure 2 shows the HRGC-(CH4-NCI)-MS (SIM) chromatogram of a typical STW effluent sample. STW effluents, representing the major source of hormonally active compounds for the aquatic environment, contain the highest concentrations of all analyzed samples (see Table 3). Endogenous steroids such as E1, aE2, and bE2 and the exogenous estrogen EE2 were determined almost unexceptionally in the lowest nanogram per liter range. BPA was found in all of the samples with a mean value of 16 ng/L. NP and OP, two stable breakdown products in the degradation process of alkylphenol polyethoxylates (APnEO), are found in a much higher range with a mean value of 200 and 22 ng/L, respectively. Table 4 shows the concentrations found in river water receiving STW effluents. Various local STWs discharge their effluent water into a larger river like the Danube and the Iller. Thus, the concentrations measured there cannot be exclusively linked to only the nearest STW upstream. On the other side, the contamination of a small creek like the Nau and the Blau that only receive wastewater of one mainly domestic STW can easily be attributed to this effluent. Therefore Nau and Blau Creeks were sampled at their source and 1 km downstream of the first STW in order to get an idea of how a hardly burdened river is contaminated by the first municipal STW. In Figure 3, the elevated concentrations upstream and downstream of these two rivers, as compared to the first STW effluent, are illustrated. Drinking Water. In Table 5, the results of tap water samples are summarized. E1 and EE2 were found in four of VOL. 35, NO. 15, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. EI-MS and NCI-MS spectra of 17r-ethinylestradiol and bisphenol A. the samples with a mean value of 400 and 350 pg/L, respectively; aE2 could only be determined in one sample at 300 pg/L, while bE2 was found in five samples with a mean value of 700 pg/L. In contrast to steroids, alkylphenols and BPA were found in all of the drinking water samples. The technical mixture NP and OP were determined with a mean value of 8.0 and 2.0 ng/L, respectively; BPA was determined with a mean value of 1.1 ng/L. No specific difference in contamination levels between tap water stemming from River Danube groundwater or Lake Constance surface water could be ascertained.
TABLE 3. Concentrations in STW Effluentsa n> LOD LOD
min
4-nonylphenol 4-tert-octylphenol bisphenol A
0.05 15 25 0.05 15 2.2 0.04 15 4.8
estrone 17R-estradiol 17β-estradiol 17R-ethinylestradiol
0.10 0.15 0.15 0.10
a
15 13 14 14
0.35 0.15 0.15 0.1
max 770 73 47 18 4.5 5.2 8.9
median mean
RSD (%)
111 14 10
110 96 62
1.5 0.5 0.4 0.7
199 22 16
3.4 140 1.0 120 0.9 140 1.4 50
Concentrations in ng/L; n ) 16 samples.
TABLE 4. Concentrations in River Watera n> LOD LOD
min
4-nonylphenol 4-tert-octylphenol bisphenol A
0.05 0.05 0.04
31 31 31
6.7 0.8 0.5
estrone 17R-estradiol 17β-estradiol 17R-ethinylestradiol
0.10 0.15 0.15 0.10
29 8 14 15
0.10 0.15 0.15 0.10
a
max 134 54 14 4.1 2.0 3.6 5.1
median mean 23 3.8 3.8 0.40 0.40 0.30 0.40
32 7.3 4.7
RSD (%) 72 52 81
0.70 125 0.60 70 0.60 34 0.80 52
Concentrations in ng/L; n ) 31 samples.
Discussion
FIGURE 2. HRGC-(NCI)-MS (SIM) chromatogram: TIC of a 1-L STW effluent sample (10-mL aliquot). u, unknown compound; 2, change of SIM tracks. 3204
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In this work, a very sensitive method for the determination of phenols and phenol analogue compounds such as estrogens was used to determine endocrine-disrupting chemicals in STW effluent, surface water, and drinking water in southern Germany. This method allows the fortification of water volumes up to 5 L by solid-phase extraction on LiChrolut EN. A quantitative derivatization to pentafluorobenzoyl esters allowed very sensitive detection of absolute femtogram amounts by HRGC-ECD and (CH4-NCI)-MS. Despite the complex matrix that STW effluent or surface water represent, in this study only some selected compounds
FIGURE 3. Comparison of concentrations upstream and downstream of the first STW in two creeks (average of both sampling sites).
TABLE 5. Concentrations in Drinking Watera n> LOD LOD
min
4-nonylphenol 4-tert-octylphenol bisphenol A
0.05 0.04 0.02
10 10 10
2.50 16 0.20 4.9 0.50 2.0
6.6 1.8 1.1
8.0 2.0 1.1
estrone 17R-estradiol 17β-estradiol 17R-ethinylestradiol
0.05 0.10 0.10 0.05
4 1 5 4
0.20 0.30 0.20 0.15
0.40 0.30 0.30 0.35
0.40 0.30 0.70 0.35
a
max
0.60 0.30 2.1 0.50
median
RSD mean (%) 58 94 52
Concentrations in ng/L; n ) 10 samples.
were investigated in order to demonstrate the effectiveness of the method. An extension for the determination of other endocrine-disrupting phenols can easily be achieved. These are, among others, cosmetic preservatives and antioxidants such as alkyl-p-hydroxybenzoates (“parabens”, e.g., methyl-, ethyl-, propyl-, and butylparabene), tert-butylhydroxyanisole (BHA), disinfectants and preservatives such as hydroxybiphenyls (e.g., 2- and 4-hydroxybiphenyl), and other bisphenols (e.g., BPAP, BPE, BPP, and BPZ). All these compounds can be equally converted to their pentafluorobenzoyl esters and are highly sensitive for ECD or NCI-MS detection. In STW discharges, the investigated steroids were found in the lower nanogram per liter range with mean concentrations of 2.5 ng/L E1, 1 ng/L aE2 and bE2, and 1.5 ng/L EE2. This correlates widely with the results of previous investigations in our lab (15) and other work groups (10-14, 24, 25). Differences in the concentrations up to a factor of 10-20 for E1, bE2, or EE2 may be caused by different weather conditions (sunshine and dryness, dilution by rain, temperature) in the sampling period, differences in pass time through the treatment process, state of the art of the sewage treatment plant, and composition of influent water. All STW samples were collected before 10 a.m. The low concentrations may be attributed to the fact that the “morning toilet” has not reached the effluent yet. It can be assumed that aE2, which is not an estrogen excreted by humans, can only stem from livestock breeding. NP and OP were found in all STW samples at mean concentrations of 200 and 22 ng/L, respectively. These results indicate that the nonionic surfactants NPnEO and OPnEO are still widely used in industrial and household cleaners (below 5% according to a recommendation of the European Community). The import of textiles to Germany from countries where APnEO is still widely used may also be a significant source. Nevertheless, a decrease up to a 10-fold
as compared to investigations in the early 1980s and 1990s is noticeable when concentrations at the microgram per liter level were spilled into the aquatic environment and led to a decrease of fish, for example, in Switzerland (4, 23). On the basis of a voluntary phase out of APnEOs in industrial surfactants in Europe, alkylphenol and APnEO concentrations are likely to decrease, but further investigations in river water and sediment are necessary to monitor and confirm these predictions in the future. BPA, frequently used as a monomer in epoxy resins and polycarbonates, was present in all STW samples with a mean value of 16 ng/L. It has been well-documented that the polymerization reactions in production are to a certain degree incomplete, and thus free monomer can be recovered from these plastic products, e.g., lacquer-coated cans (26). The effluent water of the three investigated STWs was diluted approximately 100-fold by the respective river or creek. For example, the Danube River in Ulm has a mean flow of 100 m3/s and receives about 1 m3/s STW effluent. The concentrations in the seven monitored rivers and creeks indicate that only dilution by a factor of 2-6 occurs and that all rivers already contain a certain steady load of steroids and phenols from upstream. Whether all STW effluents contribute additionally to a rising total burden of a river from source to end or degradation processes lead to a saturation or threshold value for each compound is grounds for further research. Nevertheless, a local rise of a river’s load with estrogenic active chemicals downstream is shown. If the concentrations found may contribute or lead to endocrine-disrupting effects in fish or other aquatic species cannot be said, for no wildlife studies have been conducted in these rivers. Furthermore, it was shown that these compounds can frequently be detected in drinking water samples, stemming from groundwater supplies of bank filtration of the Danube River and the surface water of Lake Constance. Steroidal estrogens and phenolic compounds could be found in the lower picogram per liter range and the lower nanogram per liter range, respectively. Obviously, the applied standard preparation for tap water is not able to remove these compounds completely. Apart from the direct entry of leachate from surface water or leaks in sewage channels, the contamination of groundwater may also be a result of fertilization in agriculture with sewage sludge. Comparing the concentrations of steroids with the daily intake dose, for example, of oral birth control pills, several thousand liters of drinking water would have to be consumed per day to reach these doses. Analogous to this, the daily intake of BPA with drinking water may be negligible as compared to the intake by foodstuff that has been in contact with polycarbonate packaging. Nevertheless, controversial discussion about BPA and its threshold values (e.g., 3 mg/kg migration in foodstuff allowed in the European Community) will continue. But ongoing research, surveying, and monitoring is essential in order to maintain or even improve the quality of surface water and drinking water in Europe (30). Another interesting aspect of our survey appears to be arising: NP, OP, and BPA were unexceptionally present as contaminants in all the samples. They were even present in blank samples of purified water (bidistilled or reverse osmosis) but mostly below the respective LOQs. Regarding the western European output figures of 130 000 t of APnEO (28) and 560 500 t of BPA in 1995 (29), the question arises whether these compounds are not already spread over the global aquatic environment and can be detected at least in picogram per liter amounts, e.g., in the Mediterranian Sea or the Atlantic Ocean. This matter is currently being investigated in our lab. If this suspicion can be confirmed, these “man-made chemicals” may already have reached the status of ubiquity. VOL. 35, NO. 15, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Acknowledgments The financial support for this work (H.M.K.) by the “Fonds der Chemischen Industrie” is acknowledged.
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Received for review February 5, 2001. Revised manuscript received May 7, 2001. Accepted May 14, 2001. ES010034M
