Phenolic xenoestrogens in surface water, sediments, and sewage sludge from Baden-Württemberg, south-west Germany

Environmental Pollution 115 (2001) 291±301

www.elsevier.com/locate/envpol

Phenolic xenoestrogens in surface water, sediments, and sewage sludge from Baden-WuÈrttemberg, south-west Germany U. Bolz *, H. Hagenmaier, W. KoÈrner Institute of Organic Chemistry, University of TuÈbingen, Auf der Morgenstelle 18, D-72076 TuÈbingen, Germany Received 8 September 2000; accepted 5 January 2001

``Capsule'': Nine phenolic chemicals that mimic estrogens were found in surface water, sediments and sewage sludge. Abstract Nine structurally di€erent phenolic chemicals, which have been reported to mimic estrogen e€ects, were determined in various aquatic environmental compartments. Twenty-three water samples from ®ve streams and rivers showed levels up to 458 ng/l for 4nonylphenol (4NP), 189 ng/l for 4-t-octylphenol (4tOP), 272 ng/l for bisphenol A (BPA) and 47 ng/l for 2-hydroxybiphenyl (2OHBiP). Elevated levels of these compounds in a stream with a high load of e‚uents of sewage treatment plants (STPs), compared to a brook free of sewage, identi®ed STPs as major sources. With a similar order, 4NP (10±259 mg/kg dry matter), 4tOP (<0.5±8 mg/kg), BPA (<0.5±15 mg/kg), and 2OHBiP (2±69 mg/kg) were also detected regularly in riverine sediment (n=11). Levels in sewage sludge were one order of magnitude higher than in sediments. 4-Hydroxybiphenyl and 4-chloro-3-methylphenol were found predominantly in sludge and sediment in the lower ppb range. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: 4-Nonylphenol; Bisphenol A; 2-hydroxybiphenyl; 4-chloro-3-methylphenol; GC/MS

1. Introduction Up to now more than 50 non-steroidal anthropogenic chemicals are known to mimic the e€ects of the natural estrogen 17b-estradiol. Xenoestrogens with widely different chemical structures have been identi®ed by in vitro (Soto et al., 1994, 1995; Jobling et al., 1995; Klotz et al., 1996; Olea et al., 1996; KoÈrner et al., 1998; Sonnenschein and Soto, 1998) and in some cases by in vivo studies (Doods and Lawson, 1936; Bitman and Cecil, 1970; Jobling et al., 1996; Gimeno et al., 1996; Nimrod and Benson, 1996; Nagel et al., 1997; Milligan et al., 1998; Christiansen et al., 2000). Many xenoestrogens including those investigated in this work possess a phenolic group. Because of their widespread application as industrial chemicals, often in aqueous solution, phenolic xenoestrogens are expected to end up primarily in the aquatic environment via sewage while the routes for phytoestrogens as natural compounds are mainly different. Recent discoveries of an increase of plasma * Corresponding author. Tel.: +49-7071-2976-219; fax: +49-7071292099. E-mail address: ulrike.bolz@uni- tuebingen.de (U. Bolz).

vitellogenin levels in wild male ®sh in rivers downstream of municipal sewage treatment plants (STPs) in the UK and the USA (Harries et al., 1996, 1997; Folmar et al., 1996) and in caged ®sh held in diluted e‚uents (Purdom et al., 1994; Harries et al., 1999; Larsson et al., 1999) indicate that STP e‚uents represent a major route for the release of biologically relevant levels of estrogenic active substances into the aquatic environment. Already during sewage treatment a partition from the aqueous phase to the particulate phase takes place besides biodegradation. Estrogenically active substances in rivers are expected to be distributed in a similar way between surface water and sediment. In particular, the ubiquitous occurrence and persistence of p-alkylphenols in all aquatic environmental matrices has been documented (for review see Thiele et al., 1997; Bennie, 1999) with the highest levels being found in sediments and sewage sludge. Interest in these biodegradation products of alkylphenol polyethoxylates began already in the 1980s when their acute toxicity to aquatic organisms became well known (McLeese et al., 1980). The discovery of the estrogenic activity of p-nonylphenol by chance (Soto et al., 1991) revived the discussion of their distribution in the environment. Only little data exists on the occurrence

0269-7491/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(01)00100-2

292

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

of other phenolic xenoestrogens such as bisphenol A (Fromme et al., 1999; Nakada et al., 1999; Lee and Peart, 2000), 2-hydroxybiphenyl, 4-hydroxybiphenyl, 4-chloro-3-methylphenol, 4-chloro-2-methylphenol, and 2-t-butyl-4-methylphenol in aquatic samples and in particular in sediments and sewage sludge. Thus, the objective of this study was to get an overview on their occurrence in the di€erent aquatic matrices. Although we already established a GC/MS method for the simultaneous quantitative determination of these phenolic xenoestrogens in sewage and surface water (Bolz et al., 2000), the development of a sample preparation method for solid samples was still necessary. To ensure its applicability on sediment and sewage sludge and to garantuee reproducible ®ndings of all substances, di€erent validation experiments had to be carried out. In this study, we analyzed surface water and sediments from di€erent streams and rivers in the state Baden-WuÈrttemberg, South West Germany. For the selection of sampling sites, special emphasis was placed on the possible in¯uence of discharges of municiple STPs on the accumulation of phenolic xenoestrogens in rivers and especially in small streams. Two small streams, one which receives a large amount of STP e‚uents and the other which is free of sewage e‚uents, were compared thoroughly by repeated sampling in 1998 and 1999. Further, several sludge samples of a major municipal STP were analyzed. 2. Experimental 2.1. Chemicals and materials A stock solution of all reference standards in a concentration of 100 mg/ml was prepared in methanol: 4-toctylphenol (4tOP) >90% purity, techn. 4-nonylphenol (4NP) with 85% content of p-isomers, bisphenol A (BPA) 97%, 4-hydroxybiphenyl (4OHBiP) >98%, 2hydroxybiphenyl (2OHBiP) >98%, 3-t-butyl-4-hydroxyanisole >98% (all obtained from Fluka, Buchs, Switzerland), 4-chloro-3-methylphenol (4Cl3MP) 99%, 4-chloro-2-methylphenol (4Cl2MP) 97%, and 2-tbutyl-4-methylphenol (2tB4MP) 99% (all purchased from Aldrich, Steinheim, Germany). The corresponding working standard solutions were obtained by dilution of the stock solution with methanol. D10-biphenyl 98% isotope purity (Cambridge Isotope Laboratories, purchased from Promochem, Wesel, Germany), used as internal standard for quanti®cation, was dissolved separately in methanol to a concentration of 120 mg/ml. All solvents (Promochem, Wesel, Germany) were of nanograde/picograde purity. NaCl (analytical grade) was treated at 400 C for 4 h and stored in a glass bottle. HCl conc., H2SO4 conc. and NaOH (all pro analysis) were purchased from Merck (Darmstadt,

Germany). The methylation reagent phenyltrimethylammoniumhydroxide was obtained as 0.1 M solution in methanol from Fluka (Buchs, Switzerland). For solid phase extraction, cartridges of 200 mg of the crosslinked polystyrene divinylbenzene copolymer phase ENV+ (6 ml, IST, Mid Glamorgan, UK purchased from Separtis, Grenzach-Wyhlen, Germany) were used. Silanized glass wool was obtained from Mallinckrodt Baker (Griesheim, Germany). 2.2. Sample preparation Surface water (1998 and 1999) was mainly taken as spot check while ®lling up brown glass bottles. Further, composite samples of the streams KoÈrsch and KraÈhenbach were taken over eight days in June 1998 (250 ml/day). The samples were stored at 4 C for up to 28 days until extraction. Sediments were collected between 1996 and 1999 from the river banks (0±4 cm). The sampling was carried out at one spot or over a distance of some meters by taking and mixing several closed cores. Sediment samples were sieved through a 1-mm sieve, freeze-dried, ground and stored in brown glass bottles at room temperature until extraction. The sampling points of surface water and sediment which were collected from various streams and rivers in Baden-WuÈrttemberg, south-west Germany, are characterized in Fig. 1. Sewage sludge samples were taken from the municipal sewage plant SteinhaÈule, Neu-Ulm, Germany, on three di€erent days in 1998 and 1999. The sludge samples were freeze-dried, ground (<1 mm) and stored in brown glass bottles.

Fig. 1. Map of sampling locations at the streams and rivers in southwest Germany (Danube=Donau).

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

2.3. Extraction and clean up For surface water solid phase extraction (SPE) was applied as a quick extraction method. First, the samples (1 l) were adjusted to room temperature, then, after pH adjustment to pH 2±3 (H2SO4 conc.) and addition of 5 ml methanol, small portions of NaCl were added to the water sample until a conductivity equal to that of a pure aqueous 0.5% NaCl solution was achieved. Silanized glass wool was added to the top of the solid phase column to prevent clogging by suspended particles during extraction. The solid phase ENV+ was conditioned with 6 ml acetone, 10 ml methanol and 5 ml distilled water (pH 2). The adsorption was performed at a ¯ow rate of 10±15 ml/min, after which the column was washed with 6 ml distilled water (pH 2) and dried under nitrogen (purity 4.6, Messer, Griesheim, Germany). The elution was performed with 22.5 ml acetone. The eluate was evaporated to a ®nal volume of 0.5 ml under a gentle stream of nitrogen. The extraction and clean up of solid samples was carried out as follows (Fig. 2): 10 g of sediment and 3 g

293

of sewage sludge, respectively, were transferred into a paper thimble (pre-extracted for at least 3 h with acetone) of a Soxhlet apparatus and were extracted with 90 ml of a mixture of methanol:diethylether (10:1, v/v) and 0.1% (v/v) HCl conc. for 24 h. The volume of the extract obtained was reduced to 3±5 ml using a rotary evaporator and diluted to 50 ml with n-hexane. This solution was transferred into a 100 ml separatory funnel and extracted, for 2 min, each with three times 20 ml of a 2 M NaOH solution. NaCl was added if necessary for better phase separation. Emulsions were broken by centrifugation (Labofuge, Haeraeus Christ) at 4000 min 1 for 10 min. For further puri®cation the n-hexane phase and the combined aqueous phases were treated separately. The aqueous phase was cooled on ice and acidi®ed with HCl conc. to pH 2±3 and, after equilibration to room temperature, adsorbed on 200 mg ENV+-phase as described above for water samples. The obtained eluate was evaporated also to a ®nal volume of 0.5 ml. The n-hexane phase did not require further puri®cation and was directly evaporated to a ®nal volume of 0.5 ml (sediment) or 5 ml (sludge). 2.4. Validation of the preparation method for solid samples

Fig. 2. Extraction and clean-up procedure for the eight phenolic xenoestrogens.

Two spiking experiments were performed, one to investigate the possible losses during the clean up procedure and one to determine the recoveries after application of the whole extraction and clean up method on a spiked sediment: (1) three replicates of 50 ml n-hexane, spiked with de®ned amounts of the nine phenolic xenoestrogens (about 1 mg per compound) were subjected to the clean up procedure and ®nally analyzed; and (2) three aliquots of a sediment sample (Braunsel, collected at 10 June 1999) were spiked (equivalent to about 140 ng/g dry matter for each compound), extracted, puri®ed and analyzed. The spiking procedure was performed as follows: 400 ml of the working standard (1:10 dilution) were dissolved in about 300 ml methanol and the solution was added to 28 g of the sediment sample. After shaking of the mixture for 15 min to obtain an even distribution of the compounds, methanol was gently removed with a rotary evaporator. The loss of phenolic compounds with the methanol vapour was determined to be less than 5%. Before analysis, the spiked sediment was stored one month at room temperature in the dark to allow aging. To calculate the recoveries, three non-spiked aliquots of the sediment sample were analyzed in parallel. Two di€erent extraction media were compared with regard to eciency and reproducibility of the extraction procedure: Aliquots of the same sewage sludge sample (STP SteinhaÈule, 12, March 1998) were extracted with a mixture of methanol/diethylether (10:1, v/v) and 0.1% (v/v) HCl conc. (n=4), and with a mixture of

294

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

n-hexane/acetone/diethylether (5:5:1, v/v) and 0.1% (v/v) HCl conc. (n=3), puri®ed and analyzed. 2.5. Determination of the loss of ignition To remove the residual water, samples were dried to a constant weight at 105 C. Then, samples were calcined at 550 C until weight constancy. The di€erence of the two weights gave the loss of ignition. 2.6. Analysis by HRGC/LRMS Aliquots of 20±50 ml of the extracts were methylated with 80±50 ml of phenyltrimethylammonium-hydroxide (0.1 M solution in methanol) at room temperature, after which 10 ml of the D10-biphenyl solution were added as internal standard. The phenolic xenoestrogens were analyzed by HRGC/ LRMS using a HP 5890 Series II gas chromatograph directly coupled to a HP 5972 A mass selective detector. The mass spectrometer was operated in the selected ion monitoring (SIM) mode to detect the methylether of the phenolic compounds. A 15 m DB-XLB fused silica capillary column with 0.25 mm inner diameter and 0.25 mm ®lm thickness (J&W Scienti®c Products, KoÈln, Germany) was used with helium as carrier gas (purity 4.6, Messer, Griesheim, Germany) at a ¯ow rate of 1.16 ml/ min. Splitless injection (1 ml of the sample) was performed using a HP autosampler with the injection port at 240 C. The temperature of the GC/MS transfer line was 290 C; the oven temperature program was as follows: 80 C for 1 min, 7 C min 1 to 180 C, 12 C min 1 to 240 C, 20 C min 1 to 300 C, 300 C for 3 min. The monitored ions of the methylether used for quanti®cation and con®rmation of the nine phenolic

xenoestrogens are shown in Table 1. Quanti®cation was performed by comparison of the peak heights of an appropriate m/z value with that of the M+ ion of D10biphenyl. Response factors relative to D10-biphenyl were determined by ®ve point calibration with derivatized standard solutions representing a concentration range between 10 pg and 5 ng absolute. For the quanti®cation of 4-nonylphenol two major peaks of the complex pattern of peaks were selected and compared with those of the technical standard (Bolz et al., 2000). The instrumental limits of detection (LOD: signal to noise ratio of 3:1) and the instrumental limits of quanti®cation (LOQ) representing the lower limits of the linear range of determination, were established for each compound separately (Bolz et al., 2000). For the di€erent samples the methodical LODs and LOQs of the phenolic xenoestrogens were as follows: surface water (LODs: <10±50 ng/l, LOQs: 10±50 ng/l), sediment (LODs: 40.5 mg/kg, LOQs: 0.6±2 mg/kg) and sewage sludge (LODs: <0.5±19 mg/kg, LOQs: 0.6±35 mg/kg). 3. Results and discussion 3.1. Validation of the sample preparation method for solid samples Since chromatography on silica was not ecient as puri®cation step before HRGC/LRMS analysis we applied a liquid-liquid separation with an alkaline aqueous solution as clean up, an approach which is widely used for separation of phenols and other weak organic acids from neutral and basic compounds. To get information on the partitioning of the investigated phenolic xenoestrogens between n-hexane and the 2 M NaOH

Table 1 m/z Values used for quanti®cation and con®rmation of the methylethers of the phenolic xenoestrogens in aqueous and solid environmental samplesa m/z Quanti®cation

4-Nonylphenol (4NP) 4-t-Octylphenol (4tOP) Bisphenol A (BPA) 2-Hydroxybiphenyl (2OHBiP) 4-Hydroxybiphenyl (4OHBiP) 4-Chloro-3-methylphenol (4Cl3MP) 4-Chloro-2-methylphenol (4Cl2MP) 2-t-Butyl-4-methylphenol (2tB4MP) 3-t-Butyl-4-hydroxyanisole (3tB4OHA)

149d 149 241 169 169 156 156 163 179

m/z Con®rmation

121 121 256 184 184 158 158 135 151

Recoveries of the preparation method for solid samples Clean upb

Extraction+clean upc

1007e 1104e 9810f 989f 12310f 978f 928f 1016e ±

719 7021 566 605 1304 6723 6013 5829 ±

a Recoveries ( RSD) determined after performance of the clean up with spiked n-hexane aliquots and after extraction and puri®cation of three aliquots of a spiked and aged sediment sample. b Aliquots of 50 ml n-hexane (n=3) spiked with 1 mg of each compound. c Aliquots of a spiked (140 ng/g of each compound) and aged sediment (n=3). d Quanti®cation of the sum of 4-nonylphenol isomers was performed by calculation the mean of two major peaks. e Sum of aqueous and n-hexane phase. f Only detectable in the aqueous phase.

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

solution during funnel separation, the clean up procedure was applied to aliquots of n-hexane spiked with these compounds. BPA, 4OHBiP, 2OHBiP, 4Cl3MP, and 4Cl2MP were found quantitatively in the solid phase extract of the aqueous phase as expected for phenolic compounds (Table 1). However, 4NP, 4tOP, and 2tB4MP were found in the hexane phase to the extent of 87, 75 and 88%, respectively. The lipophilic character and the resulting poor phenate formation of the latter three substances may explain their presence in the organic phase. The lipophilic character is supported for 4NP and 4tOP by the high log KOW>4 (Ahel and Giger, 1993) and for 2tB4MP by the possible shielding of the phenolic group by the bulky t-butyl group in o-position. Extention of the extraction time to 310 min did not change the distribution between the two fractions. The persistence of these three substances in the n-hexane phase required that both phases had to be analyzed and quanti®ed. The recoveries of the triple analysis determined as the sum of the recoveries in the aqueous and the n-hexane phase are presented in Table 1. Recoveries between 92 and 123% with RSDs below 10% proved that there is no substantial loss of the substances after performance of the funnel separation with 2 M NaOH solution. When purifying real sample extracts, the partitioning of 4NP and 4tOP between the two phases showed some variation. Matrix compounds were suspected to be responsible for this fact. Table 1 also shows the recoveries determined after Soxhlet extraction and application of the clean up procedure for three replicates of the spiked and aged sediment sample. The recoveries of eight phenolic compounds exceeded 56% and the analytical precision of the triple analysis was satisfactory with RSDs below 29%. Although especially newer extraction techniques (e.g. supercritical ¯uid extraction: SFE) achieve higher recoveries for 4NP (Lee and Peart, 1995; Kreisselmeier

295

and DuÈrbeck, 1997; Lin et al. 1999), the recoveries of our spiking experiment represent overall reasonable results. Kreisselmeier and DuÈrbeck (1997) demonstrated that despite the use of SFE the recovery of 4NP was reduced from 90% as determined in samples recently spiked to 55% in samples which had been aged for four months. In contrast to our spiking level of 140 mg/kg, much higher spiking levels of 5±90 mg/kg were used in these studies. 3tB4OHA could not be determined in any spiked sediment sample, presumably as a result of its easy chemical oxidation. Since even traces of metals or iron salts lead to the loss of its activity (Reynolds, 1997), it would be surprising to detect 3tB4OHA in sediment or sludge samples. Therefore, in comparison with the SPE method for aqueous samples, the sample preparation method for solid matrices is only applicable for eight of the nine phenolic xenoestrogens. However, using the ENV+ solid phase and eluting with acetone, the recovery experiments for aqueous samples showed relatively low recoveries for 3tB4OHA (52%) while the recoveries of all other eight compounds were between 89 and 109% (Bolz et al., 2000). In Fig. 3 the results of the replicate independent analyses of a non-spiked sewage sludge sample after application of two di€erent extraction media are compared with regard to the eciency of extraction. The concentrations of 4NP, 4tOP and BPA after extraction with methanol/diethylether (10:1, v/v)+0.1% (v/v) HCl were considerably higher than those of the extraction with nhexane/acetone/diethylether (5:5:1, v/v)+0.1% (v/v) HCl. Therefore, the extraction medium methanol/ diethylether (10:1, v/v)+0.1% (v/v) HCl was selected for subsequent analyses. The RSDs of the results of both replicate extraction series were between 2 and 34%. These reproducible ®ndings proved the suitability of the analytical method also for dicult matrices such as sewage sludge.

Fig. 3. Two di€erent extraction media were applied on aliquots of a sewage sludge sample (municipal STP SteinhaÈule, Neu-Ulm, collected on 12 March 1998) to optimize the extraction eciency. The results of the extraction with n-hexane/acetone/diethylether+HCl are given as percentage of the results of the extraction with methanol/diethylether+HCl. The concentrations of the detected phenolic xenoestrogens are given in mg/kg dm ( RSD) dry matter.

296

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

3.2. Determination of phenolic xenoestrogens in environmental samples 3.2.1. Surface water 4NP, 4tOP, BPA and 2OHBiP were detectable in nearly all 23 water samples collected from various streams in South West Germany. 4NP showed on average the highest concentrations of all nine phenolic xenoestrogens with up to 458 ng/l. In comparison to 4tOP and BPA, which were detected in 20 and in all 23 samples respectively, 4NP was only detectable in 16 samples 4tOP and BPA reached peak levels of 189 and 272 ng/l. 2OHBiP was detected in 14 samples, the levels did not exceed 47 ng/l. Low levels of 3tB4OHA and the p-chlorocresols were found only in water samples of the small stream KoÈrsch. 4OHBiP and 2tB4MP were not detectable at all (LODs: 10 ng/l). The repeated sampling of water of the two streams KoÈrsch and KraÈhenbach over a period of one year showed a clear di€erence in the concentrations (compare medians in Table 2). The medians of all detected phenolic xenoestrogens in the KoÈrsch (¯ow: 57 Mio m3/ a), which receives e‚uents of six STPs (20 Mio m3/a), were obviously higher than those of the KraÈhenbach, which is free of discharges of STPs. Especially, the maximum levels of BPA, 2OHBiP and 3tB4OHA of the KraÈhenbach were clearly lower than those of the KoÈrsch. In the e‚uent of the STP Stuttgart-MoÈhringen, the biggest of the six STPs discharging into the KoÈrsch, 81 ng/l of BPA was found in August 1998 (KoÈrner et al., 1999). This e‚uent level can explain the median BPA concentration of 72 ng/l in the KoÈrsch because the discharges are diluted by a factor of only three on an average and in times of drought are nearly undiluted. Therefore, discharges of STPs have to be suspected to be the major route for the release of these latter three phenolic substances into streams and rivers. Although the medians of 4NP and 4tOP were clearly higher in the KoÈrsch, the highest level of 4NP of all investigated water samples (485 ng/l) was measured in the KraÈhenbach on 16 January 1999. Wood-cutting activities performed at the stream banks in those days

may be the possible source for 4NP since NP polyethoxylates (NPEO) are still used as lubricant additives and thus were likely to be carried into the stream by contaminated wood shavings which were seen on the water surface at the sampling site. In summary, the monitoring at the two streams KoÈrsch and KraÈhenbach over one year revealed that, beside the continuous entrance of phenolic xenoestrogens via discharges of STPs, there are also other sources for their release into streams. Di€use as well as selective sources have much stronger impacts on the levels of pollutants in small streams such as the KoÈrsch, the KraÈhenbach, the Erms and the Echaz, which do not have a large body of water and thus a smaller dilution than in large rivers. The analytical results indicated that beside 4NP and 4tOP, BPA and 2OHBiP are regularly occurring pollutants at lower levels in small streams as well as in big rivers such as the Danube. In two studies from Germany and the Netherlands comparable concentrations of between <0.1 and 410 ng/l were found for BPA (Hendriks et al., 1994; Fromme et al., 1999). Also the regular occurrence of 2OHBiP in German rivers was con®rmed (Franke et al., 1995; Ternes et al., 1998). 3.2.2. Sediment As in surface water, 4NP, 4tOP, BPA and 2OHBiP were found in nearly all sediment samples. In contrast to the water samples, 4OHBiP and 4Cl3MP were also detected in the majority of the 11 sediment samples. The concentrations of the eight phenolic xenoestrogens in sediments of the streams Braunsel, KraÈhenbach, Echaz, KoÈrsch and Sulzbach and of the rivers Neckar and Danube ranged from n.d. to 259 mg/kg dry matter (dm, see Table 3). The highest levels of the eight compounds were measured for 4NP, as in surface water samples. In the sediment of the Braunsel, which, like the KraÈhenbach is a slightly contaminated stream without discharges of STPs, phenolic xenoestrogens were detected indicating again di€use sources. However, the levels in both streams were on an average lower than in sediments of the other streams and rivers receiving e‚uents of STPs, especially for 4NP. This result together with

Table 2 Concentrations (median) in ng/l of phenolic xenoestrogens in surface water of streams and rivers in Baden-WuÈrttemberg, south-west Germanya No. of samples KoÈrsch (side A)

8

KraÈhenbach

9

Danube (side A±C) Erms (side A/B) Neckar (side A)

3 2 1

a

n.d., Not detectable.

4NP

4tOP

BPA

2OHBiP

n.d.±164 (66.7) n.d.±485 (25.1) 56±233 n.d./74 73

19.5±189 (31.4) n.d.±155 (<20) <20±163 n.d./n.d. <20

<50±272 (72.1) <50±59 (<50) <50±86 <50/<50 <50

n.d.±47 (23.9) n.d.±23 (n.d.) <20 <20/<20 <20

4OHBiP

4Cl3MP

4Cl2MP

2tB4MP

3tB4OHA

n.d. (n.d.) n.d. (n.d.) n.d. n.d. n.d.

n.d.±<10 (n.d.) n.d. (n.d.) n.d. n.d. n.d.

n.d.±<10 (n.d.) n.d. (n.d.) n.d. n.d. n.d.

n.d. (n.d.) n.d. (n.d.) n.d. n.d. n.d.

n.d±35 (11.7) n.d. (n.d.) n.d. n.d. n.d.

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

297

Table 3 Concentrations [mg/kg dry matter] of phenolic xenoestrogens in sediments of streams and rivers in Baden-WuÈrttemberg, south-west Germany and in sludge of the municipal STP SteinhaÈule, Ulm/Neu-Ulm, Germanya No. of samples KoÈrsch (side A±C) KraÈhenbach Sulzbach Neckar (side B/A) Echaz Danube (side C) Braunsel Sewage Sludge a

3 2 1 2 1 1 1 3

4NP

4tOP

BPA

36±203 20/10 39 78/259 43 94 10 2517±3675

4±5 3/n.d. 6 5/8 4 9 1 77±201

3±7 n.d./2 4 6/15 10 n.d. 6 70±770

2OHBiP

4OHBiP

4Cl3MP

4Cl2MP

2tB4MP

Loss of ignition (%)

2±3 2/3 2 30/69 48 28 2 63±172

4±6 2/2 5 4/6 3 2 2 n.d.±12

1±15 1/n.d 2 n.d. n.d. 2 1 14±40

n.d. n.d. n.d. n.d. n.d. 2 n.d. n.d.

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

±/5.2/9.1 ± 5.6 8.1/14 6.6 8.2 6.8 64/67/±

n.d., Not detectable; ±, not determined.

the analyses of the sediment samples of the river Neckar collected both 500 m upstream (side B) and 500 m downstream (side A) of the discharge of the municipal STP TuÈbingen indicates that STP e‚uents are sources for the accumulation of 4NP in the sediments of streams and rivers. For direct comparison of the sediment analyses of the Neckar, the concentrations were calculated on the basis of the content of loss of ignition. Fig. 4 shows a twofold increase in the concentrations of 4NP downstream of the STP discharge. In contrast, the higher concentrations of 2OHBiP and BPA downstream of the STP TuÈbingen could also be the result of the limited analytical precision (RSDs of 16±34%), determined for a sewage sludge sample (compare Fig. 3). Considering the lower log KOW 3.4 of BPA (Staples et al, 1998) than that of 4NP, accumulation of BPA in sediment was not to be expected. The concentrations of BPA between 1 and 190 mg/kg dm recently measured in German and Japanese sediments (Fromme et al., 1999; Nakada et al. 1999) are comparable with our results (n.d.±15 mg/kg dm). Although the majority of the sampling points of the streams and rivers investigated in this work are located in urbanized and industrialized areas and downstream

of STP discharges (Echaz, KoÈrsch, Neckar B, Sulzbach), the concentrations of 4NP (10±259 mg/kg dm) and 4tOP (n.d.±9 mg/kg dm) were in some cases considerably lower than those (4NP up to 37 800 mg/kg dm, 4tOP up to 8220 mg/kg dm) found in English, and North American rivers in the Great Lakes area (Bennie et al., 1997; Bennet and Metcalf, 1998, 2000; Lye et al., 1999; Hale et al., 2000). Even higher concentrations of the two alkylphenols (up to 72 000 mg/kg dm) were present on an average in Canadian and Korean lakes (Bennie et al., 1997; Bennett and Metcalf, 1998; Khim et al., 1999a). Studies in Switzerland and Germany showed similarly high concentrations for 4NP (up to 13 100 mg/kg) as in English and American rivers (Ahel et al., 1994b; Zellner and Kalbfus, 1997; Fromme et al., 1999). In contrast to these ®ndings there are also data of sediment analyses which are comparable to our low results. In the sediments of the German river Main only a maximum concentration of 700 mg/kg dm (median <50 mg/kg) was found (Trapp et al., 1992). De Voogt et al. (1997) detected concentrations between 0.1 and 17 mg/kg dm for 4NP and between n.d. and 2 mg/kg dm for 4tOP in riverine, estuarine and marine sediment located around the North Sea. Although Khim et al. (1999b) found

Fig. 4. Comparison of the analyses of phenolic xenoestrogens in sediments of the Neckar upstream and downstream of the discharge of the STP TuÈbingen. The concentrations are given in mg/kg loss of ignition.

298

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

higher 4NP concentrations (113±3890 mg/kg) in sediments from the southeast coast of Korea than in our study, they observed a comparable relationship between the levels of 4NP and the level of 4tOP and BPA, which were on an average 20±110-fold lower than those of 4NP. The sediment concentrations of 4tOP (<0.5±9 mg/ kg dm) and BPA (<0.5±5 mg/kg dm) detected in our work lay on an average 2±190-fold lower than those of 4NP (10±259 mg/kg). 3.2.3. Sewage sludge The concentration of the detected phenolic xenoestrogens of n.d. up to 3675 mg/kg dm were one order of magnitude higher in the sewage sludge samples than in sediment samples (Table 3). The sludge samples collected at three di€erent dates showed more or less similar concentrations for all identi®ed phenolic xenoestrogens. The SIM-traces of BPA, 2OHBiP and 4OHBiP obtained of the sludge sample, collected on 6 July 1999 are shown in Fig. 5. The m/z values used for quanti®cation and con®rmation by HRGC/LRMS enabled unequivocal identi®cation of the three compounds. The presence of 4NP, 4tOP, BPA, 2OHBiP, and 4Cl3MP in the sewage sludge and their recent detection in the raw sewage of the same STP (KoÈrner et al., 2000) con®rms that STPs are possible sources for their release into the aquatic environment when adsorption to sewage sludge and biodegradation are incomplete. The amount of release of these compounds

into streams and rivers via e‚uents is dependent on their input and the subsequent puri®cation eciency of the STP. In the e‚uent of the STP SteinhaÈule, only 4NP, 4tOP, BPA, and low levels of 2OHBiP were detectable (KoÈrner et al., 2000) indicating a good puri®cation eciency for 2OHBiP and 4Cl3MP. In the absence of data for the other phenolic xenoestrogens in sewage sludge or sediments, only the results for 4NP, 4tOP and BPA can be compared with those of other studies. The concentrations of 4NP between 2500 and 3700 mg/kg detected in this work in the sludge samples of the STP SteinhaÈule seem to be very low, but it has to be taken into account that the sludge was not anaerobically stabilized. Wahlberg et al. (1990) detected a fourfold increase in the concentration of 4NP upon anaerobic stabilization of a sewage sludge. During anaerobic sludge stabilization the primary metabolites of 4NP polyethoxylates are degraded to 4NP (Ahel et al., 1994a). Also lower 4NP concentrations in undigested sewage sludge were found by Lee and Peart (1999) when analyzing samples from Canadian pulp and paper mills. Analyzing more than 149 sewage sludge samples from the Western part of Germany from 1987 to 1989 (median: 83 400 mg/kg dm, 90%-percentile: 264 000 mg/kg dm) and then again 101 sewage sludge samples from the state Rheinland-Pfalz in Germany from 1994 to 1995 (median: 4600 mg/kg dm, 90%percentile: 24 800 mg/kg dm), Jobst (1995, 1998) noted a signi®cant decrease of the levels of 4NP. The rather low

Fig. 5. SIM traces of the m/z values for quanti®cation and con®rmation of BPA, 2OHBiP and 4OHBiP, detected in a sewage sludge sample (municipal STP SteinhaÈule, Neu-Ulm, collected at 6 July 1999) by HRGC/LRMS.

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

4NP concentration in the sewage sludge of the STP SteinhaÈule and Jobst results indicate that the voluntary agreement of the German detergent industry from 1986 to exclude alkylphenol polyethoxylates from all commercial formulations intended for domestic applications has led to a consequent drop in the concentrations of 4NP polyethoxylates and their metabolite 4NP in wastewater of domestic origin. In other countries such as Canada (up to 368 000 mg/kg dm) and Taiwan (up to 244 000 mg/kg dm), much higher concentrations of 4NP have been recently found (Bennet and Metcalfe, 1998; Lin et al., 1999). Also higher levels of BPA, ranging from 33 to 36 700 mg/kg, were detected in sludge samples of Canadian STPs (Lee and Peart, 2000) than in the samples of the STP SteinhaÈule (70±770 mg/kg). In contrast, Fromme et al. (1999) found comparable concentrations between 4 and 1360 mg/kg in 31 German sewage sludge samples. 3.2.4. Possible relevance for aquatic wildlife To assess the relevance of the measured concentrations for aquatic wildlife, it is not sucient to consider only the concentrations of the individual substances, but it is absolutely imperative to get knowledge on the endocrine potential, the e€ects of mixtures and the persistence of all estrogenically active substances. The estrogenic potency of the investigated phenolic xenoestrogens is considerably lower than that of the natural estrogens. 104±107-Fold higher concentrations than 17bestradiol are necessary to exert the same de®ned e€ect. The levels of 4NP, 4tOP and BPA found in the present study are below the e€ect level for juvenile freshwater ®sh (1 mg/l for 4NP; Ash®eld et al., 1998) and the predicted no-e€ect concentration of 64 mg/l for BPA (Staples et al., 1998). However, it has to be considered that mixtures of xenoestrogens act in an additive manner, both alone and together with natural and synthetic estrogens (Soto et al., 1994; Routledge et al., 1998, Thorpe et al., 2000), resulting in a total estrogenic activity which may cause adverse e€ects to aquatic organisms. Levels of natural estrogens in the lower ng/l range as previously published in other studies (Snyder et al., 1999; Ternes et al., 1999; Belfroid et al. 1999) were found in all investigated streams and rivers (KoÈrner et al., submitted). While the e€ects of some waterborne phenolic xenoestrogens (mainly p-alkylphenols and derivatives) to ®sh have been well investigated there is hardly any data on e€ects of sediment bound xenoestrogens to benthic invertebrates. However, very recent laboratory experiments with the marine netted whelk Hinia reticulata exposed for one month to freshwater sediments covered with arti®cial seawater have revealed ®rst e€ects of feminization at sediment concentrations of 10 mg/kg dm for BPA and 50 mg/kg dm for 4tOP. Similar e€ects have been observed with sediment samples from two

299

German rivers (J. Oehlmann, personal communication). In our study, BPA concentrations in two of the sediment samples were at that level. BPA and 4tOP exposure via the water resulted also in a feminization of further prosobranch species, like for example Nuclla lapillus, Marisa cornuarietis and Potamoyprgus antipodarum, with LOEC values of 1 mg/l (Oehlmann et al., 2000). 4. Conclusion In this study we have sucessfully applied a GC/MS method recently developed for aqueous samples to determine phenolic xenoestrogens in surface water, sediment and sewage sludge. A sample preparation method for solid matrices was established employing Soxhlet extraction with subsequent clean up, which enables the simultaneous determination of eight di€erent phenolic xenoestrogens in sediments and sewage sludge, in particular of compounds like BPA, 2OHBiP, 4OHBiP, 4Cl3MP, 4Cl2MP and 2tB4MP which have scarcely been investigated in sediment and sludge. The investigation of surface water and sediment of di€erent streams and rivers in Baden-WuÈrttemberg, south-west Germany and of sewage sludge of a major STP showed at least that four of the target substances were found regularly in all three matrices with following order 4NP>4tOPBPA>2OHBiP. Substances like 4OHBiP and 4Cl3MP were predominantly detected in solid samples only. The concentrations of 4NP and 4tOP measured in this work were on an average lower than those found in the existing German studies or in studies from other countries indicating ®rst positive e€ects of the voluntary agreement of the German detergent industry of 1986. However, our data also shows that the current release of p-alkylphenols, BPA, and 2OHBiP into streams and rivers occurs mainly via discharges of STPs leading to elevated levels in surface water and sediment. Small streams receiving a large amount of STP e‚uents are particularly subjected to intensi®ed pollution. Information on the time trend of BPA and 2OHBiP levels in the aquatic environment is not yet possible. Because of the obviously widespread occurrence of BPA and 2OHBiP in the aquatic environment and in sewage sludge, although at low levels at present, these widely used chemicals should be measured together with p-alkylphenols in future monitoring programs, since e€ects of BPA to certain species of invertebrates may be possible at levels in surface water and sediment in the low ppb range. Although in this study 4Cl3MP was only found in a few samples and at low levels, 4Cl3MP should be included in the target list of estrogenically active substances for monitoring of aquatic environmental matrices, because data of Castillo et al. (1999) veri®ed its occurrence at levels of 3.4 mg/l in tannery

300

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301

wastewater while we recently found less than 1 mg/l in the in¯uent of a major municipal STP in south-west Germany (KoÈrner et al., 2000). At present in Germany only 400 t of 4Cl3MP are used but more than 1600 t are exported (UBA, 1996). Acknowledgements The support by the fellowship program of the German Environmental Foundation (Deutsche Bundesstiftung Umwelt) is gratefully acknowledged. We like to express our thanks to Dr. Rita Triebskorn and her coworkers, Zoological Institute, University of TuÈbingen, for their assistance in the sampling of surface water and sediment. References Ahel, M., Giger, W., 1993. Partitioning of alkylphenols and alkylphenol polyethoxylates between water and organic solvents. Chemosphere 26 (8), 1471±1478. Ahel, M., Giger, W., Koch, M., 1994a. Behaviour of alkylphenol polyethoxylates surfactants in the aquatic environment Ð I. Occurence and transformation in sewage treatment. Water Research 28 (5), 1131±1142. Ahel, M., Giger, W., Scha€ner, C., 1994b. Behaviour of alkylphenol polyethoxylates surfactants in the aquatic environment Ð II. Occurrence and transformation in rivers. Water Research 28 (5), 1143± 1152. Ash®eld, L.A., Pottinger, T.G., Sumpter, J.P., 1998. Exposure of female juvenile rainbow trout to alkylphenolic compounds results in modi®cations to growth and ovosomatic index. Environ. Toxicol. Chem. 17 (4), 679±686. Belfroid, A.C., Van der Horst, A., Vethaak, A.D., Schafer, 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. Science of the Total Environment 225 (1±2), 101±108. Bennett, E.R., Metcalfe, C.D., 1998. Distribution of alkylphenol compounds in Great Lakes sediments, United States and Canada. Environmental Toxicology and Chemistry 17 (7), 1230±1235. Bennett, E.R., Metcalfe, C.D., 2000. Distribution of degradation products of alkylphenol ethoxylates near sewage treatment plants in the lower Great Lakes, North America. Environmental Toxicology and Chemistry 19 (4), 784±792. Bennie, D.T., 1999. Review of the environmental occurrence of alkylphenols and alkylphenols ethoxylates. Water Quality Research Journal of Canada 34 (1), 79±122. Bennie, D.T., Sullivan, C.A., Lee, H.-B., Peart, T.E., Maguire, R.J., 1997. Occurrence of alkylphenols and alkylphenol mono- and diethoxylates in natural waters of the Laurentian Great Lakes basin and the upper St Lawrence River. Science of the Total Environment 193 (3), 263±275. Bitman, J., Cecil, H.C., 1970. Estrogenic activity of DDT analogs and polychlorinated biphenyls. Journal of Agriculture and Food Chemistry 18 (6), 1108±1112. Bolz, U., KoÈrner, W., Hagenmaier, H., 2000. Development and valildation of a GC/MS method for the determination of phenolic xenoestrogens in aquatic samples. Chemosphere 40 (9±11), 929±935. Castillo, M., Alonso, M.C., Riu, J., Barcelo, D., 1999. Identi®cation of polar, ionic, and highly water soluble organic pollutants in untreated industrial wastewaters. Environmental Science and Technology 33 (8), 1300±1306.

Christiansen, L.B., Pedersen, K.L., Pedersen, S.N., Korsgaard, B., Bjerregaard, P., 2000. In vivo comparison of xenoestrogens using rainbow trout vitellogenin induction as a screening system. Environmental Toxicology and Chemistry 19 (7), 1867±1874. De Voogt, P., De Beer, K., Van der Wielen, F., 1997. Determination of alkylphenol ethoxylates in industrial and environmental samples. Trends in Analytical Chemistry 16 (10), 584±595. Doods, E.C., Lawson, W., 1936. Synthetic estrogenic agents without the phenanthrene nucleus. Nature 137, 996. Folmar, L.C., Denslow, N.D., Rao, V., Chow, M., Crain, D.A., Enblom, J., Marcino, J., Guilette, L.J., 1996. Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant. Environmental Health Perspectives 104 (10), 1096±1101. Franke, S., Hildebrandt, S., Schwarzbauer, J., Link, M., Francke, W., 1995. Organic compounds as contaminants of the Elbe River and its tributaries. Part II: GC/MS screening for contaminants of the Elbe water. Fresenius. Journal of Analytical Chemistry 353, 39±49. Fromme, H., KuÈchler, T., MuÈller, J., Wenzel A., 1999. Monitoring of estrogenic substances in the environment. In: Kettrup, A., Schramm, K.-W. (Eds.), Proceedings of SECOTOX 99, Fifth European Conference on Ecotoxicology and Environmental Safety, 15± 17 MaÈrz, MuÈnchen. GSF-Bericht 02/99. GSF Forschungszentrum fuÈr Umwelt und Gesundheit. Gimeno, S., Gerritsen, A., Bowmer, T., Komen, H., 1996. Feminization of male carp. Nature 384 (6606), 221±222. Hale, R.C., Smith, C.L., de Fur, P.O., Harvey, E., Bush, E.O., 2000. Nonylphenols in sediments and e‚uents associated with diverse wastewater outfalls. Environmental Toxicology and Chemistry 19 (4), 946±952. Harries, J.E., Janbakhsh, A., Jobling, S., Matthiessen, P., Sumpter, J.P., Tyler, C.R., 1999. Estrogenic potency of e‚uent from two sewage treatment works in the United Kingdom. Environmental Toxicology and Chemistry 18 (5), 932±937. Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, P., Sumpter, J.P., Tylor, T., Zaman, N., 1997. Estrogenic activity in ®ve UK rivers detected by measurement of vitellogenesis in caged male trout. Environmental Toxicology and Chemistry 16 (3), 534± 542. Harries, J.E., Sheahan, D.A., Jobling, S., Matthiessen, P., Neall, P., Routledge, E.J., Rycroft, R., Sumpter, J.P., Tylor, T., 1996. A survey of estrogenic activity in United Kingdom inland waters. Environmental Toxicology and Chemistry 15 (11), 1993±2002. Hendriks, A.J., Mass-Diepeveen, J.L., Noordsij, A., Van der Grag, M.A., 1994. Monitoring response of XAD-concentrated water in the Rhine Delta: A major part of toxic compounds remains undenti®ed. Water Research 8 (3), 581±598. Jobling, S., Reynolds, T., White, R., Parker, M.G., Sumpter, J.P., 1995. A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environmental Health Perspectives 103 (6), 582±587. Jobling, S., Sheahan, D., Osborne, J.A., Matthiessen, P., Sumpter, J.P., 1996. Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals. Environmental Toxicology and Chemistry 15 (2), 194±202. Jobst, H., 1995. Chlorophenols and nonylphenols in sewage sludges. Part I: Occurence in sewage sludges of Western German treatment plants from 1987 to 1989. Acta Hydrochimica et Hydrobiologica 23 (1), 20±25. Jobst, H., 1998. Chlorophenols and nonylphenols in sewage sludges. Part II: Did contents of pentachlorophenol and nonylphenols reduce? Acta Hydrochimica et Hydrobiologica 26 (6), 344±348. Khim, J.S., Villeneuve, D.L., Kannan, K., Lee, K.T., Snyder, S.A., Koh, C.H., Giesy, J.P., 1999a. Alkylphenols, polycyclic aromatic hydrocarbons, and organochlorines in sediment from Lake Shihwa, Korea: Instrumental and bioanalytical characterization. Environmental Toxicology and Chemistry 18 (11), 2424±2432.

U. Bolz et al. / Environmental Pollution 115 (2001) 291±301 Khim, J.S., Kannan, K., Villeneuve, D.L., Koh, C.H., Giesy, J.P., 1999b. Characterization and distribution of trace organic contaminants in sediment from Masan Bay, Korea. 1. Instrumental analysis. Environmental Toxicology and Chemistry 33 (23), 4199±4205. KoÈrner, W., Spengler, P., Bolz, U., Hagenmaier, H., Metzger, J., 1999. Monitoring of estrogenic substances in sewage plant e‚uents by biological and chemical analysis. Organohalogen Compounds 42, 29±32. KoÈrner, W., Hanf, V., Schuller, W., Bartsch, H., Zwirner, M., Hagenmaier, H., 1998. Validation and application of a rapid in vitro assay for assessing the estrogenic potency of halogenated phenolic chemicals. Chemosphere 37 (9±12), 2395±2407. KoÈrner, W., Bolz, U., SuÈûmuth, W., Hiller, G., Schuller, W., Hanf, V., Hagenmaier, H., 2000. Input/outputbalances of estrogenic active compounds in a major municipal sewage plant in Germany. Chemosphere 40 (9), 1131±1143. KoÈrner, W., Bolz, U., Triebskorn, R., Schwaiger, J., Negele, R.-D., Marx, A., Hagenmaier, H. Steroid analysis and xenosteroid potential in two small streams in Southwest Germany. Journal of Aquatic Ecosystem Stress and Recovery, submitted. Klotz, D.M., Beckman, B.S., Hill, S.M., McLachlan, J.A., Walters, M.R., Arnold, S.F., 1996. Identi®cation of environmental chemicals with estrogenic activity using a combination of in vitro assays. Environmental Health Perspectives 104 (10), 1084±1089. Kreisselmeier, A., DuÈrbeck, H.W., 1997. Determination of alkylphenols, alkylphenolethoxylates and linear alkylbenzenesulfonates in sediments by accelerated solvent extraction and supercritical ¯uid extraction. Journal of Chromatography A775 (1±2), 187±196. Larsson, D.G.J., Adolfsson-Erici, M., Parkkonen, J., Pettersson, M., Berg, A.H., Olsson, P.-E., FoÈrlin, L., 1999. Ethinyloestradiol Ð an undesired ®sh contraceptive? Aquatic Toxicology 45 (2±3), 91±97. Lee, H.-B., Peart, T.E., 1995. Determination of 4-nonylphenol in e‚uent and sludge from sewage treatment plant. Analytical Chemistry 67 (13), 1976±1980. Lee, H.-B., Peart, T.E., 1999. Occurrence of nonylphenol ethoxylates and their metabolites in Canadian pulp and paper mill e‚uents and sludge. Water Quality Research Journal of Canada 34 (4), 633±652. Lee, H.-B., Peart, T.E., 2000. Bisphenol A contamination in Canadian municipal and industrial wastewater and sludge samples. Water Quality Research Journal of Canada 35 (2), 283±298. Lin, J.-G., Arunkumar, R., Liu, C.-H., 1999. Eciency of supercritical ¯uid extraction for determining 4-nonylphenol in municipal sewage sludge. Journal of Chromatography A840 (1), 71±79. Lye, C.M., Frid, C.L.J., Gill, M.E., Cooper, D.W., Jones, D.M., 1999. Estrogenic alkylphenols in ®sh tissues, sediments, and waters from the UK Tyne and Tees estuaries. Environmental Science and Technology 33 (7), 1009±1014. McLeese, D.W., Zittko, V., Metcalfe, C.D., Sergeant, D.B., 1980. Lethality of aminocarb and the components of the aminocarb formulation to juvenile atlantic salomon, marine invertebrates and a freshwater clam. Chemosphere 9 (2), 79±82. Milligan, S.R., Balasubramanian, A.V., Kalita, J.C., 1998. Relative potency of xenobiotic estrogens in an acute in vivo mammalian assay. Environmental Health Perspectives 106 (1), 23±26. Nagel, S.C., vom Saal, F.S., Thayer, K.A., Dhar, M.G., Boechler, M., Welshons, W.V., 1997. Relative binding anity-serum modi®ed access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol-A and octylphenol. Environmental Health Perspectives 105 (1), 70±76. Nakada, N., Isobe, T., Nishiyama, H., Okuda, K., Tsutsumi, S., Yamada, J., Kumata, H., Takada, H., 1999. Broad-spectrum analysis of endocrine disruptors in environmental samples. BunsekiKagaku 48 (6), 535±547. Nimrod, A.C., Benson, W.H., 1996. Estrogenic responses to xenobiotics in channel cat®sh (Ictalurus punctatus). Marine Environmental Research 42 (1±4), 155±160.

301

Oehlmann, J., Schulte-Oehlmann, U., Tillmann, M., Markert, B., 2000. E€ects of endocrine disruptors on prosobranch snails (Mollusca: Gastropoda) in the laboratory. Part I: Bisphenol A and octylphenol as xeno-estrogens. Ecotoxicology 9 (6), 383±397. Olea, N., Pulgar, R., PeÂrez, P., Olea-Serrano, F., Rivas, A., NovilloFertrell, A., Pedraza, V., Soto, A.M., Sonnenschein, C., 1996. Estrogenicity of resin-based composites and sealants used in dentistry. Environmental Health Perspectives 104 (3), 298±305. Purdom, C.E., Hardiman, P.A., Bye, V.J., Eno, N.C., Tyler, C.R., Sumpter, J.P., 1994. Estrogenic e€ects of e‚uents from sewage treatment works. Chemistry and Ecology 8 (4), 275±285. Reynolds, J.E.F., 1997. Martindale. The Extra Pharmacopoeia. Royal Pharmaceutical Society, London, UK. Routledge, E.J., Sheahan, D., Desbrow, C., Brighty, G.C., Waldock, M., Sumpter, J.P., 1998. Identi®cation of estrogenic chemicals in STW e‚uent. 2. In vivo responses in trout and roach. Environmental Science and Technology 32 (11), 1559±1565. Sonnenschein, C., Soto, A.M., 1998. An updated review of environmental estrogen and androgen mimics and antagonists. Journal of Steroid Biochemistry and Molecular Biologie 65 (1), 143±150. Soto, A.M., Justicia, H., Wray, J.W., Sonnenschein, C., 1991. pNonylphenol: an estrogenic xenobiotic released from ``modi®ed'' polystyrene. Environmental Health Perspectives 92 (May), 167±173. Soto, A.M., Chung, K.L., Sonnenschein, C., 1994. The pesticides endosulfan, toxaphene, and dieldrin have estrogenic e€ects on human estrogen-sensitive cells. Environmental Health Perspectives 102 (4), 380± 383. Soto, A.M., Sonnenschein, C., Chung, K.L., Fernandez, M.F., Olea, N., Olea-Serrano, M.F., 1995. The E-Screen assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environmental Health Perspectives 103 (Suppl. 7), 113±122. Snyder, S.A., Keith, T.L., Verbrugge, D.A., Snyder, E.M., Gross, T.S., Kannan, K., Giesy, J.P., 1999. Analytical methods for detection of selected estrogenic compounds in aqueous mixtures. Environmental Science and Technology 33 (16), 2814±2820. Staples, C.A., Dorn, P.B., Klecka, G.M., Block, S.T., Harris, L.R., 1998. A review of the environmental fate, e€ects, and exposures of bisphenol A. Chemosphere 36 (10), 2149±2173. Ternes, T.A., Stumpf, M., Schuppert, B., Haberer, K., 1998. Simultaneous determination of antiseptics and acid drugs in sewage and river water. Vom Wasser 90, 295±309. Ternes, T.A., Stumpf, M., Mueller, J., Haberer, K., Wilken, R.D., Servos, M., 1999. Behavior and occurrence of estrogens in municipal sewage treatment plants Ð I. Investigations in Germany, Canada and Brazil. Science of the Total Environment 225 (1±2), 81±90. Thiele, B., GuÈnther, K., Schwuger, M.J., 1997. Alkylphenol ethoxylates: Trace analysis and environmental behavior. Chemical Reviews 97 (8), 3247± 3272. Thorpe, K.L., Hetheridge, M.J., Hutchinson, T.H., Scholze, M., Sumpter, J.P., Tyler, C.R., 2000. Mixtures of environmental chemicals have additive e€ects in vivo. 10th Annual Meeting of SETAC Europe, Brighton, U.K., May 21±25, Abstract 4G/006, pp. 44. Trapp, S., BruÈggemann, R., Kalbfus, W., Frey, S., 1992. Organische und anorganische Schadsto€e im Main. Gwf Wasser/Abwasser 133 (10), 495±504. UBA, Umweltbundesamt (Ed.), 1996. Bewertung der UmweltgefaÈhrlichkeit ausgewaÈhlter Altsto€e durch das Umweltbundesamt. (Teil II UBA-Texte 38/96), Berlin, Germany. Wahlberg, C., Renberg, L., Wideqvist, U., 1990. Determination of nonylphenol and nonylphenol ethoxylates as their penta¯uorobenzoates in water, sewage sludge and biota. Chemosphere 20 (1±2), 179±195. Zellner, A., Kalbfus, W., 1997. Belastung bayrischer GewaÈsser durch Nonylphenole. In: Bayrische Landesinstitut fuÈr Wasserwirtschaft, Institut fuÈr Wasserforschung (Ed.), Sto€e mit endokriner Wirkung im Wasser (MuÈncher BeitraÈge zur Abwasser-, Fischerei-, und Fluûbiologie: Bd. 50) R. Oldenburg Verlag MuÈnchen, Germany, pp. 55±64