Environ. Sci. Technol. 2000, 34, 5076-5083
Estrogenicity Determination in Sewage Treatment Plants and Surface Waters from the Catalonian Area (NE Spain) M O N T S E R R A T S O L EÄ , * , † MARIA J. LO Ä PEZ DE ALDA,† MONTSERRAT CASTILLO,† CINTA PORTE,† KNUD LADEGAARD-PEDERSEN,‡ AND D A M I AÅ B A R C E L O Ä † Environmental Chemistry Department, IIQAB-CSIC, Jordi Girona, 18-2608034 Barcelona, Spain, and Institute of Biology, Odense University, 5230, Odense M, Denmark
Polyethoxylated nonylphenol (NPEOx) surfactants and nonylphenol (NP) as their major degradation product as well as some synthetic and natural estrogens and progestogens have been reported to be present in freshwater systems, mainly at the vicinity of urban discharges and sewage treatment plants (STPs), at levels high enough to exhort estrogenicity to wildlife. To determine both presence and effects of such compounds in two tributaries of the Llobregat river (NE Spain), water samples and carp, Cyprinus carpio, were collected from selected sites along a transect, for chemical and biological determinations, respectively. Also influent and effluent water from several STPs, discharging into these rivers, was collected for its chemical characterization. NP and NPEO were determined by solid-phase extraction (SPE) followed by liquid chromatography-mass spectrometry (LC-MS). Representative estrogens, both natural (estradiol, estriol, estrone) and synthetic (ethynyl estradiol, mestranol, diethylstilbestrol), progestogens (norethindrone, levonorgestrel), and the natural hormone progesterone were determined by offline SPE followed by LC-diode array detection (DAD)-MS. High levels of NP were encountered in all water samples (up to 600 µg/L), whereas only a few samples gave quantifiable levels of estrogens and progestogens (ng/L range). Western blot analysis of male carp plasma vitellogenin (VTG), using a polyclonal antibody raised in the cyprinid Koi carp, detected this protein in all samples, the VTG increase being more evident at the vicinity of the treatment plants. A certain correlation was also found between NP in water and VTG induction in fish (r ) 0.75).
Introduction An increasing broad spectrum of compounds, both natural and synthetic, is reported to exhibit varying degrees of estrogenicity (1). The persistent nature and lipophilicity of some of them, or their degradation products, are an alert as to the possible effects to wildlife and human reproduction * Corresponding author phone: 34 93 4006100; fax: 34 93 2045904; e-mail:
[email protected]. † IIQAB-CSIC. ‡ Odense University. 5076
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(2). Among such compounds, estrogens and progestogens, both natural and synthetic, as well as the nonionic surfactants alkylphenol polyethoxylates (APEs) and their major degradation product, the persistent nonylphenol (NP), deserve particular attention: the steroids for displaying the highest estrogenic capacities and the surfactants because of their massive use. Estrogenicity of APEs and other “weakestrogens” is reported several orders of magnitude lower than that of the natural estradiol (3). APEs not only are present in detergents but also are found in paints, herbicides, pesticides, and many other formulated products (e.g. cosmetics). More than 500 000 tons of APEs are produced annually worldwide, 60% of which ends up in the aquatic environment, and because of their reported toxicity to aquatic life, some northern European countries have voluntarily banned their use in household cleaning products while restrictions on industrial applications are set to follow in the year 2000 (4). Exogenous estrogenic (e.g. 17R-ethynyl estradiol, diethylstilbestrol (DES), and mestranol) and progestational (e.g. levonorgestrel, norethindrone) chemicals are largely used for treatment of certain hormonal disorders (e.g. menopause) and cancers and in birth control pills. Consequently, while APEs have mainly an industrial origin, human excretion is thought to be the principal source of estrogens and progestogens in the aquatic environment. In the case of the NPEOs, incomplete degradation in the STPs leads to increasing levels of NP, which is more toxic than the parent compound. The natural and synthetic steroids are excreted mostly in a less active conjugated form; however, deconjugation by microorganisms during water treatment originates the more potent parent compound (5, 6). Consequently, the presence of such derived compounds will be more relevant at the vicinity of the STPs. In fact, the first field evidence of sexual abnormalities in wild fish was given in the mid-1980s by anglers from the U.K. who observed a higher hermaphroditism incidence in roach (Rutilus rutilus) near STPs. This determined the initiation of a nationwide investigation measuring the biological effects of compounds discharged by the STPs to freshwater fish. A widely accepted measure of estrogenicity is the determination of vitellogenin (VTG) in male fish plasma (7, 8). VTG is a yolk egg precursor, and its synthesis, in female fish, is regulated by estradiol circulating levels in plasma. In males, as a consequence of exposure to substances that mimic the natural estradiol, VTG can also be synthesized but as a redundant protein in this gender; the diversion of vital proteins or lipids to VTG formation will have adverse consequences. Several laboratory experiments support this: waterborne exposure, as the most environmentally realistic situation, indicated a direct relationship among (1) E2 dosage, VTG synthesis, and egg production (9); (2) E2, estrone dosage, and inhibition of testicular growth in fathead minnows (Pimaphales promelas) (10), and (3) alkylphenol (AP) exposure with VTG synthesis as well as inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) (11). Field evidence of a direct relationship between VTG synthesis and diminished reproductive health has been observed in flounder (Platichthys flesus) (12). Also in the cyprinid roach (R. rutilus) VTG increase correlated with the incidence of intersexuality, a widespread phenomena described for this species in the U.K. (13). The implications of biochemical changes in a physiological performance or even into a population level reinforce the adequacy of VTG as an early warning biomarker of estrogenic exposure. Further support for VTG synthesis as an indication of xenoexposure in fish has been obtained from in vitro studies after experimental exposure of rainbow trout 10.1021/es991335n CCC: $19.00
2000 American Chemical Society Published on Web 11/10/2000
FIGURE 1. Map indicating in numbers where water and fish samples were collected. [ STP Calaf, 9 STP Piera, f STP Igualada, and 2 STP Manresa. hepatocites to APs (14, 15) or in vivo studies in relation to estrogens exposure in rainbow trout and roach (16). Most field studies applying VTG as a biomarker have used caged rainbow trout as sentinel species (17-22). Fewer studies have been conducted with wild cyprinids, namely carp (23, 24) in the U.S.A. or roach (13) in the U.K. This can be attributed to the fact that VTG response in cyprinids is far less evident than in salmonids (15, 25). Most field studies have related a diminished VTG response at increasing water effluent dilution or distance from the outfall, but only a few could relate the in vivo response to specific compounds, thus the contribution of APs and natural and synthetic estrogens was usually speculative. For this, the main objective of the preset study was to determine the presence of xenoestrogens in two tributaries of the Llobregat river that supply water to the city of Barcelona by using advanced analytical methods such as solid-phase extraction followed by liquid chromatography-mass spectrometry (LCMS) and observe if these compounds had any effect on the natural fish populations inhabiting these waters. Carp was selected because physical and chemical properties of these waters could not support trout, and besides, carp are the most abundant and distributed fish in them. To our knowledge this is the first report on estrogenicity determination in Spanish waters and one of the few studies that correlates levels of nonylphenol with observed estrogenicity in carp, since most of the studies are reported in trout.
Experimental Section. Chemicals and Reagents. Polyethoxylated nonylphenol and nonylphenol were purchased from Kao Corporation (Barcelona, Spain). Pure standards of both natural and synthetic estrogens and progestogens were purchased as powders from Sigma (St. Louis, MO). HPLC-grade solvents acetonitrile, methanol, and water were purchased from Merck (Darmstadt, Germany). Acetic and sulfuric acid proanalysis grade were purchased from Panreac (Barcelona, Spain) and Merck, respectively. Water Collection. Between April and June 1999, water samples from the influent and effluent from selected STPs were collected as 24-h composite samples. STP Piera, STP Calaf, and STP Igualada discharge into the Anoia tributary, and STP Manresa discharges into the Cardener tributary. Grab samples were also taken from the same river stretches where fish were collected (Figure 1). Samples were collected in Pyrex borosilicate amber glass containers. Each bottle was rinsed with tap water and with high-purity water prior to sample addition. Sample preservation was accomplished by
storing the bottles at 4 °C immediately after sampling. Extraction is carried out as soon as possible in order to avoid addition of chemical preservatives. However, if extraction does not take place within 48 h after collection, sulfuric acid is added until the pH reaches 3 to prevent biological degradation. Fish Collection. Adult carp, Cyprinus carpio, were fished between April and June 1999 by DC electric pulse. At least 10 individuals were collected per site; approximately 1 mL of blood was taken from the caudal vein using a heparinized syringe, and 1 mM PMSF (phenylmethylsulfonyl fluoride) was added to the blood to avoid proteolysis. After the extractions, blood was immediately centrifuged at 1000g × 10 min, and the corresponding plasma were frozen to -80 °C for VTG analysis. Afterward, animals were killed and measured, their sex was determined, and other tissues and organs were collected for other biochemical and chemical analyses. Polyethoxylated Nonylphenol and Nonylphenol Analysis. Sample Preparation Procedures. The wastewater samples were filtered with a 0.45 µm membrane filter and allowed to equilibrate at room temperature before extraction. The experiment was performed using an automated sampler processor ASPEC XL (Automated Sample Preparation with Extraction Columns) from Gilson (Villiers-le-Bel, France). A more detailed description of the method is described elsewhere (26), and the methodology described here is part of a multistep analytical procedure. The extraction procedure was based on the use of solidphase extraction (SPE) with an octadecylsilica (C18) sorbent known as Lichrolut RP-18 (500 mg, 6 mL) from Merck. The condition step was performed by passing 7 mL of methanol followed by 3 mL of water through the cartridge at 1 mL/ min. A loading volume of 200 mL of wastewater was applied at 5 mL/min, and afterward the cartridges were dried with a Baker LSE 12G apparatus (J. T. Baker, Deventer, The Netherlands) connected to a vacuum system at -15 psi. The drying step took 20-30 min. Elution was performed by passing 2 × 5 mL of methanol at 1 mL/min, and the obtained extract was evaporated to dryness with a stream of nitrogen. The extract was reconstituted to a final volume of 1 mL in the appropriate HPLC mobile phase prior to analysis. LC-APCI-MS Analysis. Twenty microliters of the SPE extract was injected in the LC systems using the conditions described below. Separation of target analytes was accomplished using water (B) and 50% acetonitrile, 50% methanol (A) both acidified with 0.5% of acetic acid as the mobile phase and VOL. 34, NO. 24, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Scheme of the analytical procedure for the analysis of estrogens and progestogens in water samples. a Hypersil Green ENV column (150 mm × 4.6 mm i.d., 5 µm particle size) equipped with a guard column both from Shandon HPLC (Cheshire, U.K.). The mobile phase composition was 70% B during the first 10 min, then linearly decreased to 0% B in 15 min, and remained isocratic for 5 min. The flow rate was maintained at 1 mL/min. Identification and quantification of nonylphenol derivatives were performed by LC-APCI-MS using a VG Platform system from Micromass (Manchester, U.K.) equipped with a standard atmospheric pressure ionization (API) source, which can be configured for APCI or ESI. Source and probe temperatures were set at 150 and 400 °C, respectively, corona discharge voltage was maintained at 3 kV, and the cone voltage was set at 30 V. The HV lens voltage was set at 0.20 kV. Nitrogen was used as a nebulizing and drying gas at a flow rate of 10 and 300 L/h, respectively. In full scan mode the m/z range was from 100 to 400 in negative ion (NI) mode and from 70 to 1000 in positive ion (PI) mode of ionization. Characteristic peaks in PI mode at 133, 177, 271, and 291 m/z units were checked for polyethoxylated nonylphenol. In addition to this comparison, the [M + H]+ ion of NPEO4+6 (C9H19-C6H4(OCH2CH2)xOH), being that x is either four or six ethoxylate groups, was determined by the characteristic m/z ions at 397 ( 44 and 485 ( 44, respectively. More details about the LC-APCI-MS determination of NPEO are described elsewhere (27). The presence of some degradation products was checked in NI mode with characteristic peaks at m/z 5078
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219 and 277 for nonylphenol (NP) and nonylphenol monoethoxy carboxylate (NPEC), respectively. External calibration was used for quantitation of target analytes. Estrogens and Progestogens Analysis. Sample Preparation Procedure. The overall schematic procedure is shown in Figure 2. A more detailed description of the method development and validation is described elsewhere (28). Briefly, samples were filtered through glass fiber filters as for nonionic surfactants analysis, and extraction was performed also using an ASPEC XL processor. Samples (500 mL) were percolated at 5 mL/min through previously conditioned Lichrolut RP-18 cartridges and posteriorly dried with a Baker LSE 12G apparatus. Elution was performed, in this case, by passing a total volume of 2 × 4 mL of acetonitrile dispensed with a 5-min delay between them. The extracts obtained were then evaporated under nitrogen and reconstituted with methanol to a final volume of 0.5 mL for subsequent LCDAD-MS. LC-DAD-MS Analysis. The HPLC system consisted of an HP 1100 autosampler with the volume injection set to 20 µL and an HP 1090 A LC pump both from Hewlett-Packard (Palo Alto, CA). Separation is achieved, in the first instance, on a Lichrospher 100 RP-18 column (250 × 4 mm, 5 µm) preceded by a guard column (4 × 4 mm, 5 µm) of the same packing material from Merck. Samples suspected to contain target analytes are secondarily analyzed in another column with different selectivity toward the analytes: a Lichrospher 60
TABLE 1. Recoveries and Limit of Detection (LOD) Obtained for Water Samples Using the Protocol Described in the Experimental Section compd
recovery (%) (RSD, N ) 3)
LOD (water samples)a (µg/L)
NPEO4+6b NPEC NP
102 (5) 88 (7) 91 (9)
0.20 0.08 0.15
a Concentration factor 500. b These standards of NPEO corresponded with an average of four and six ethoxy units.
RP-Select B (250 × 4 mm, 5 µm) preceded by a guard column (4 × 4 mm, 5 µm) of the same packing material from Merck. Gradient elution acetonitrile/water was used as the mobile phase. Detection was performed with a diode array detector model 1040M coupled in a series with a mass spectrometer HP 1100 MSD API-ES, all from Hewlett-Packard (Palo Alto, CA). UV chromatograms were recorded at 197, 225, and 242 nm. UV spectra from 190 to 600 nm were simultaneously recorded to aid identification through comparison with libraries created for that purpose. MS detection was performed by using two different interfaces: electrospray (ESP) and atmospheric pressure chemical ionization (APCI). Both ESP and APCI, in the positive ion mode of operation, were used for the group of progestogens: ESP as a first option to be used with the Lichrospher 100 RP-18 column, and APCI as a second option to be used with the Lichrospher 60 RP-Select B column for confirmation of previously determined positive samples. For the group of estrogens ESP was in the negative ion mode in both instances since these compounds were not detected with the APCI interface. Chromatograms were recorded under timescheduled selected ion monitoring conditions. Nitrogen was used as a nebulizing and drying gas. Some other experimental parameters are summarized in Figure 2. The external standard method was used for quantitation. Quantitation. Polyethoxylated Nonylphenol and Nonylphenol. The identification of target compounds was done in a full scan mode by matching the retention time and mass spectrum with authentic standards. Full scan positive ionization mode acquisition with m/z range from 100 to 1000 was used for the screening of nonylphenol polyethoxylated surfactants, while full scan negative ionization mode acquisition with m/z range from 100 to 400 was used for the screening of degradation products (NPEC, NP, and final quantification were performed in a selected ion monitoring mode (SIM) using external calibration.). A series of injections of target compounds in the concentration range from 0.05 to 50 mg/L was used to obtain the calibration equations. Calibration curves (concentration vs peak area) were generated using linear regression analysis and over the established concentration range gave good fits (r 2 values >0.990). The recoveries (percent of standard added to the sample recovered during extraction and cleanup) and reproducibility (relative standard deviation for triplicate analysis) of the method were determined by spiking experiment. The limits of detection of target compounds in aqueous samples (wastewater and river water) were calculated by a signal-to-noise ratio of 3 (the ratio between the intensity of signal of each compound in standard solution obtained with SIM conditions and intensity of noise), taking into account the amount of sample extracted, the volume of the extract analyzed, and recovery rate obtained from a parallel assay of a spiked sample. The recoveries and limits of detection are reported in Table 1. Estrogens and Progestogens. For quantitation the external standard method was used in all instances. 17β-
Estradiol-d2 was initially considered for its use as an internal standard but finally discarded. The coelution with its homologous nondeuterated 17β-estradiol and the tight molecular mass difference (2 uma) between these two compounds restrained its use as an internal standard with either detector (DAD or MS). The HP LC/MSD ChemStation software application was used to assist in the quantitation, based on peak areas, of standards and samples. Five-point calibration curves were constructed using a least-squares linear regression analysis from the injection of standard solutions of the mixture of all analytes at concentrations ranging from 1 to 100 µg/mL. When MS detection was accomplished in the positive ion mode of operation, with either ESP or APCI, seven-point calibration curves were constructed covering a wider range of concentrations (10 µg/mL to 25 ng/mL) as a consequence of its considerably better sensitivity. Table 2 lists the correlation coefficients (r 2) obtained for every analyte and detector. Good linearity was observed (r 2 > 0.99) except in the case of the MS detector when operating with the ESP interface in positive ion mode (r 2 > 0.94). An explanation for such low correlation coefficients could be due to the likely instability of the adducts formed between the analyte molecule and the sodium atom, which were used for quantitation in this case. Detection limits (LODs) were experimentally estimated from the injection of standard solutions serially diluted until the signal-to noise ratio (S/N) for any single analyte reached a value of three. LODs fell between 50 and 500 ng/L for DAD, between 2 and 500 ng/L for ESP-MS, and between 20 and 5000 ng/L for APCI-MS. As it can be seen, mestranol is not detected by any of the MS interfaces and modes tested, and, therefore, its simultaneous determination with the rest of the analytes considered in this study requires the use of the diode array detector in series with the MS detector. Since no certified reference materials were available, the overall method of repeatability and accuracy was determined from the analysis of six replicates of distilled water (0.5 L) spiked with a standard mixture of the analytes at 10 µg/L. Satisfactory recoveries (% R > 83) were obtained for all compounds except for diethylstilbestrol (% R ) 56). The low recovery percentage obtained for diethylstilbestrol is probably not a consequence of the extraction procedure but the result of some kind of equilibrium between the two different isomeric forms of the compound, as yet not determined, as revealed in the presence of two peaks with the same mass spectrum at different retention times and with intensities that increase and decrease in approximately reversed proportions. The overall method repeatibility was satisfactory although, as it could be expected, it was better with the UV detector (RSD < 18%) than with the MS detector (RSD < 25%). The calibration curve correlation coefficients (r 2) and LODs are reported in Table 2. Purification of Carp Vitellogenin for Antibody Production. Koi carps (Cyprinus carpio) were purchased from a local dealer in Odense, Denmark and kept in a 500 L aquaria supplied with aerated freshwater (11-14 °C) at a 12 h light: 12 h dark photo period. After an acclimation period of 1 week, and again after an additional 2 weeks, the fish were injected intraperitoneally with 5 mg/kg of 17β-estradiol dissolved in coconut oil. Prior to injections and blood samplings the fish were anaesthetized with 0.02% phenoxyethanol. After 4 weeks of exposure blood was collected from the caudal artery in heparinized tubes and protease inhibitors, and 1 mM PMSF (phenylmethylsulfonyl flouride) and 0.02% (w/w) aprotinin were added. Samples were kept on ice until the plasma was obtained by centrifugation (2500g for 15 min; 4 °C). The resulting supernatant was added along with the same amount of PMSF as above. Vitellogenin was purified by gel filtration and ion exchange chromatography. Fresh VOL. 34, NO. 24, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Calibration Curve Correlation Coefficients (r 2) and Detection Limits (LOD) (in ng/L) Obtained for Each Analyte and Detector DAD 197/242 nm 2
compound
λ (nm)
r
estriol estradiol ethinyl estradiol estrone diethylstilbestrol mestranol norethindrone levonorgestel progesterone
197 197 197 197 242 197 242 242 242
0.9996 0.9996 0.9998 0.9998 0.9995 0.9998 1.0000 0.9995 1.0000
a
225 nm 2
LOD
r
50 50 50 50 50 50 50 50 100
1.0000 0.9999 0.9999 0.9999 0.9996 1.0000 0.9999 0.9989 1.0000
ESP 2
LOD
r
100 100 100 100 100 100 100 100 100
0.9945 0.9979 0.9975 0.9988 0.9982 n.d.a 0.9484 0.9495 0.9474
APCI LOD 50 250 500 100 25 n.d.a 2 2 2
r
2
n.d.a 0.9999 0.9995 0.9997
LOD 5000 3000 3000 3000 2000 n.d.a 20 20 20
n.d. denotes not detected.
plasma from the estradiol treated fish (1 mL) was diluted in a 1:4 ratio in 50 mM Tris-HCl, pH 8.0, and immediately loaded onto a 26 × 700 mm Sephacryl S300 HR gel filtration column equilibrated with 50 mM Tris-HCl, pH 8.0; 4 °C. The sample was eluted at a flow rate of 25 mL/h, and fractions of 4.5 mL were collected. UV absorbance at 280 nm was continuously monitored. Fractions containing vitellogenin were pooled, and samples were applied to a 1 mL HiTrap Q (Pharmacia Biotech) ion exchange column equilibrated with 50 mM TrisHCl, pH 8.0, at room temperature. The sample was eluted with a linear gradient of NaCl (0.0-1.0 M) at a flow rate of 1 mL/min. UV-absorbance was continuously monitored at 280 nm, and fractions of 1 mL were collected and stored at 5 °C immediately after elution. Vitellogenin containing fractions were pooled and dialyzed against 20 mM NH4HCO3, pH 8.0, at 4 °C. The dialysate was lyophilized in a Hetovac vacuum concentrator and stored at -80 °C until used for immunization. Purification of Zebrafish Vitellin for Affinity Column Production. Zebrafish were purchased from a local dealer in Odense, Denmark and kept in 200 L aquaria supplied with aerated freshwater (27 °C) at a 12 h light:12 h dark photo period. The fish (one per L) were exposed for a month to 1 µg/L of 17β-estradiol dissolved in 96% ethanol. Water was changed twice every week. Ovaries from 20 zebrafish were dissected out and immediately frozen in liquid nitrogen. The tissue was crushed in a mortar filled with liquid nitrogen, and the resulting powder was added three times the weight of 0.9% NaCl, 1 mM PMSF, 0.02% (w/w) aprotinin. After sonication for 2 × 15 s, the sample was centrifuged for 2 h, 50 000g, 4 °C. The supernatant was immediately chromatographed by gel filtration and ion exchange chromatography as described above for carp vitellogenin. Obtained vitellin containing fractions were dialyzed and stored as described above. Immunization. Polyclonal antibodies against carp vitellogenin were raised in rabbits. Each rabbit was injected subcutaneously eight times with 0.5 mg of carp vitellogenin in 0.9% NaCl dissolved 1:1 in Freunds incomplete adjuvant. Four injections were given at 1 week intervals, and four additional injections were given at 2 week intervals. The rabbits were sacrificed and bled 2 weeks after the last injection. The blood was allowed to clot at room temperature, and the resulting antiserum was frozen at -20 °C until further purification. Purification of Antibodies. Antibodies were purified by affinity chromatography after precipitation with saturated (NH4)2SO4 as described elsewhere (29). An affinity column was prepared by coupling 5 mg of purified zebrafish vitellin to 1 g of CNBr-activated Sepharose 4B according to the recommendations from the manufacturer (Amersham Phar5080
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macia Biotech). Loading, elution, and dialysis of antibodies were performed as described in ref 29. Plasma Vitellogenin Analysis. Plasma protein content was determined by the method of Lowry et al. (30), and an equivalent amount of protein was boiled for 5 min in SDSPAGE sample buffer (31) and afterward loaded into a 7.5% polyacrylamide gel topped with 4% polyacrylamide stacking gels. The amount of protein loaded per lane was 50 µg for the field samples and 0.5 µg for the EE2 induced carp used as control. Immunoblotting. Proteins which were separated on the SDS-polyacrylamide gels were further transferred to nitrocellulose membranes (Trans-Blot, BioRad) using a transfer buffer containing 150 mM glycine in 20 mM Tris and 20% methanol (v/v). Transfer was carried out for 30 min at room temperature using a Trans-Blot semidry cell (Bio-Rad). The nitrocellulose membranes were then blocked for 30 min in Tris buffered saline pH 8.0 (TBS) which contained 0.2% Tween 20, 0.5% gelatin, and 0.1% sodium azide. The membranes were probed using 1:1000 dilution rabbit koi-carp vitellogenin polyclonal antiserum (Odense University, Denmark). Blots were incubated at room-temperature overnight and rinsed three times with TBS containing 0.2% Tween 20 and 0.5% gelatin. The membrane was incubated for 1 h with alkaline phosphatase-conjugated antirabbit IgG (Sigma Chemical Co.). An excess of secondary antibody was removed by three additional washes in 0.2% Tween 20 in TBS, and the sites of binding of the antibody were visualized by incubation with the substrates p-nitroblue tetrazolium chloride and 5-bromo4-chloro-3-indolyl phosphate. When the optimal signalbackground ratio had been achieved, rinsing the membrane in water stopped the development reaction. Western blots were semiquantitated by scanning with a densitometer PhosphoImager (Bio-Rad). Relative quantities of VTG were determined as peak areas of the bands from molecular weights ranging from 70 to 200 kDa due to the appearance of several degradation bands in the field samples. VTG protein is a very labile protein, and occurrence of degradation has been described in many fish species including carp (23, 24). Control fish were provided by a fish farm located in the Ebro Delta. A blood sample composite corresponding to a pool of four male fish injected with a single dose of 500 µg/kg EE2 for 8 days and another sample corresponding to a pool of five untreated male carp were used as positive and negative controls, respectively. Results are obtained by semiquantitation by scanning, and the absorbance of the samples is expressed as percents of the VTG protein content of the positive control. Males, which showed, by Western blot, no VTG bands using several polyclonal antibodies (AA-1, OP-1 from Biosense laboratories), gave a background absorbance of 10% of the positive control. More details characterizing
TABLE 3. Concentration of NP, NPEO, NPEC, and Estrogens in Several STPsd characteristics of treated water (distance to the main urban site)
NP (µg/L)
influent STP Piera effluent STP Piera influent STP Calaf effluent STP Calaf influent STP Igualada effluent STP Igualada
Anoia Tributary domestic 131 (10 km) 6 domestic and plastic 343 (2 km) 142 domestic, textil and tannery 40 (10 km) 289
influent STP Manresa effluent STP Manresa
domestic and rubber (1 km)
Cardener Tributary 280 n.q.c
NPE4+6 (µg/L)
NPEC (µg/L)
estriol (ng/L)
DESa (ng/L)
33 n.d.b 24 10 938 n.d.b
8 60 4 100 80 270
261 n.d.b n.d.b n.d.b 263 n.d.b
n.d.b 34 43 n.d.b n.d.b n.d.b
n.d.b n.db
0.21 n.d.b
n.d.b n.d.b
n.d.b n.d.b
a DES: diethylstilbestrol. b n.d. denotes not detected. c n.q. denotes not quantified due to the presence of interferences. a biological treatment involving activated sludge process.
d
All the plants follow
TABLE 4. Concentration of NP and NPEO and NPEC in the Receiving Waters Where Fish Were Collected weight (g)
size (cm)
physical condition (number male)
NP (µg/L)
NPEO4+6 (µg/L)
NPEC (µg/L)
plant distance (km)
site 1 (upstream) site 2 (downstream) site 3 (downstream)
678 ( 206 204 ( 44 180 ( 7.3
35 ( 4.8 22.4 ( 2.3 23.5 ( 0.7
Anoia Tributary mature (4) immature (4) immature parasited (4)
18 644 n.d.a
n.d.a 100 n.d.a
n.d.a 70 n.d.a
5 23 27
site 1 (upstream) site 2 (downstream) site 3 (Llobregat)
360 ( 68 117 ( 65 321 ( 344
29.9 ( 1.9 21.5 ( 2.4 26.4 ( 8.4
Cardener Tributary mature (5) mature (6) mature parasited (3)
51 398 42
n.d.a 20 n.d.a
n.d.a 40 n.d.a
1.5 4 8
a
n.d. denotes not detected.
the reference fish used in this study are presented elsewhere (32). Statistical Treatment. Differences between sites were tested by one-way repeated measures analysis of variance (ANOVA) followed by Bonferroni’s method, and statistically significant differences from the control were considered when p < 0.05.
Results and Discussion Chemical Analysis. High levels of NPEO4+6 and NP in the influent and effluent were determined in the four STPs studied (Table 3). One of the questions to be answered here is why the levels of NP at site 1 (upstream of the river, before treatment) of both rivers are still relatively high, 18 and 51 µg/L for Anoia and Cardener, respectively. These relatively high levels can be attributed mainly to two origins: the use of NPEO upstream of the river, and consequently NP is degraded in the STP upstream, and to an agricultural origin. High agricultural practices take place in this area, and sewage sludge is used for soil amendment. Sewage sludges in that region can contain between 100 and 500 mg/kg of NP, and with the irrigation practices NP is directed to the receiving waters and river sediments, with the subsequent background contamination. This will be reported in a future article (M. Petrovic, M. Sole´, D. Barcelo´, manuscript in preparation) which shows that river sediments forming in this regions upstream of the STPscontain between 50 and 200 µg/Kg of NP, indicating that NP has been spread out all over the area. NP and NPEC concentration increased in the effluent of the Igualada STP, where the NPEO load was more important, indicating that both degradation products are formed at the STP. NPEC increased in all the effluents of the STP discharging into the Anoia river compared to those of the influent. In the case of the Cardener tributary, NPEC is not detected in the effluent of the STP, and this is attributed to the fact that no NPEO was detected in the influent of the STP, so NPEC was not formed.
NP levels ranged from 6 to 343 µg/L in the STPs and ranged from nondetected to 644 µg/L in the receiving waters (Table 4). In sewage treated effluents, major nonylphenolic compounds are nonylphenol (NP) and nonylphenolcarboxilates (NPECs) indicating that transformation products are much more resistant to microbial degradation. NP values presented here are higher than those recently reported in the influent of the Igualada STP (from n.d. to 175 µg/L) (33). In the U.K., an extensive study reported NP levels up to 330 µg/L in STPs and up to 180 µg/L in the receiving waters for the most polluted rivers; these levels decreased to
