Animals and exposures
Ramshorn snails, M. cornuarietis (Mesogastropoda: Ampullariidae), came from our laboratory breeding stock, which was maintained at a temperature of 22 ± 1°C. The stock was derived from Aquazoo Düsseldorf (Düsseldorf, Germany) in 1991 with regular crossbreeding of wild-caught animals from Florida to avoid inbreeding. Exposure experiments were performed as 24-hr (weekends, 48-hr) semistatic renewal systems in 60-L glass aquaria (water volume, 54 L), provided with an Eheim power filter and aeration (Eheim, Deizisau, Germany). Animals were fed regularly with TetraMin (Tetra, Melle, Germany) ad libitum, supplemented with lettuce in exposure series I. In exposure series II, TetraMin was used exclusively. Tests were performed at constant temperature with an equal light:dark cycle (12:12 hr). Water parameters (pH, conductivity, temperature, nitrite, O2 concentration, and saturation) were measured twice a week per tank. Initial values were pH 7.1–7.5, 809–924 μS/cm, < 1 mg NO2 (nitrogen dioxide)/L, and 80.8–102.2% O2 saturation.
Two experimental series were conducted with an exposure to different nominal concentration ranges of BPA (Merck Schuchardt, Hohenbrunn, Germany), including a solvent control (SC, ethanol concentration: 12.5 μL/L) and a positive control [PC; 17α-ethinylestradiol (EE2; Fluka, Buchs, Germany); nominal concentration: 0.01 μg/L, only exposure series I]. The applied ethanol concentrations do not affect reproduction in M. cornuarietis, as demonstrated in extensive lab studies with androgenic (tributyltin, triphenyltin, methyl testosterone, fenarimol, letrozole) and anti-androgenic compounds (p,p′-dichlorodi-phenyldichloroethylene, cyproterone acetate, linuron) in the European Union project COMPRENDO (Comparative Research on Endocrine Disrupters).
Exposure series I
Groups of 210 sexually mature snails (shell height > 20 mm, age > 18 months) were exposed to nominal BPA concentrations of 0, 0.05, 0.1, 0.25, 0.5, and 1 μg/L for 6 months (from September to March) in charcoal-filtered tap water at 22 ± 1°C. Thirty specimens were collected for analysis at the beginning of the experiment, and from each group at monthly intervals. Initial density in the tank was 3.89 snails/L, which dropped to a maximum of 0.56 snails/L during the last month.
Exposure series II
Two replicate groups of 30 sexually mature snails (shell height sssss> 20 mm, age > 18 months) were exposed to nominal BPA concentrations of 0, 0.25, 0.5, 1, and 5 μg/L alone, and to 5 μg BPA/L in combination with a potent antiestrogen [3 μg ICI 182,780/L (Faslodex; Tocris, Ellisville, MO, USA) or 10 μg tamoxifen/L (Sigma Chemical Co., Deisenhofen, Germany)] for 5 months (February to July) in fully reconstituted water at 20 ± 1°C and 27 ± 1°C in parallel. Animals were acclimatized to exposure temperatures 2 weeks before the start of the experiment. Thirty specimens were analyzed at the beginning, and all surviving animals were analyzed at the end. Initial density in the tank was 1.11 snails/L, which dropped to 0.72–1.00 snails/L at the end of the experiment, depending on the mortality in the different groups.
Two-L water samples were taken in brown glass bottles during 24-hr cycles (at months 1, 3, and 5 for exposure series I, and at month 1 for exposure series II). Sampling began 15 min before the change of exposure media and ended 1 day later, before media were changed again (per cycle: n = 8). For extraction, solid-phase material (500 mg RP-C18 Bulk Sorbent; Separtis GmbH, Grenzahl-Wyhlen, Germany) was placed into glass cartridges, which were then conditioned by flushing with 1 × 2 mL hexane, followed by 1 × 2 mL acetone and 3 × 2 mL methanol. Cartridges were washed with 5 × 2 mL of water adjusted to pH 7. One-half liter of the water sample adjusted to pH 7 was glass-fiber filtered (< 1 μm) and spiked with bisphenol F as surrogate standard. Samples were sucked through packed glass cartridges at a flow rate of 20 mL/min. Subsequently, the solid phase was dried by a nitrogen stream for 1 hr, and analytes were eluted 4 times with 1 mL of acetone. Acetone extracts were evaporated to 200 μL by a gentle nitrogen stream.
For detection by gas chromatography–mass spectrometry (GC-MS), extracts were derivatized by adding 50 μL of the derivatization mixture [N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA)/trimethylsilylimidazole (TMSI)/dithioerytrol (DTE), 1,000 + 2 + 2, vol/vol/wt]. MSTFA and TMSI were purchased from Sigma (Deisenhofen, Germany), and DTE was obtained from Merck (Darmstadt, Germany). After 1 hr of reaction at 60°C, the solution was evaporated to dryness by a gentle nitrogen stream, and the residue was dissolved in 200 μL hexane. Finally, 100 μg mirex (Promochem, Wesel, Germany) was added to the final extract as an internal standard. For quantification, m/z = 357 and 372 were used for GC-MS detection in the SIM (selected ion monitoring) mode. A 10-point calibration was performed during the whole procedure after spiking deep groundwater with BPA in concentrations ranging from 0.01 to 5 μg/L. In each analysis series, a blank sample of deep groundwater was run in parallel.
The GC-MS system included an HP 5890 Series II coupled with an HP 5971 MS detector (Hewlett-Packard, Palo Alto, CA, USA). A Restek XTI-5 capillary column (Restek GmbH, Bad Homburg, Germany) was used at a head pressure of 85 kPa with a 3-μL splitless injector (250°C isotherm). The GC temperature program was set to a 50°C isotherm for 1.5 min, then ramped up at 20°C/min to 240°C/min, and at 1.5°C/min to 290°C/min, and finally set to a 290°C isotherm for 10 min.
Mortality, numbers of eggs, clutches, and eggs per clutch in the tanks were recorded daily. Fecundity parameters were corrected for number of females per tank by taking into consideration the number of females analyzed in monthly intervals as well as mortality data with an assumed 1:1 sex ratio. Measures of variability for produced eggs and clutches could be calculated for exposure series II (two replicates) but not for series I (one replicate).
Snails were narcotized before analysis for a minimum of 1.5 hr (2.5% MgCl2 in distilled water), and their shells were cracked and removed. The extensions of all sex organs were measured to the nearest 0.1 mm under a dissection microscope, and malformations such as oviduct ruptures or excrescences on genital and other organs were recorded.
Competitive receptor displacement experiments
Cytosolic extracts were prepared from testes or ovaries of up to 10 individuals to achieve a fresh weight of 3 g, homogenized in 9 mL ice-cold incubation buffer [20 mM Tris–HCl (pH 7.4), 250 mM sucrose, 10 mM sodium molybdate, 5 mM dithiothreitol]. Homogenates were centrifuged at 12,000× g for 12 min, and the supernatant was centrifuged a second time at 100,000× g for 60 min at 4°C. The final supernatant represented the cytosolic extract, and it was used for displacement experiments. In time-course experiments for specific [3H]-E2 and testosterone ([3H]-T), binding conditions were optimized using incubation times of 18 and 16 hr for estrogen and androgen binding, respectively, when a steady state was obtained. Radiolabeled ligands ([3H]-E2, specific activity 41.8 μCi/mmol; PerkinElmer, Rodgau-Jugesheim, Germany; and [3H]-T, 95 μCi/mmol; Amersham Biotech, Freiburg, Germany) were dissolved in 5% ethanol. Incubations were performed in duplicate using 25μL [3H]-E2 (final concentration 24 nM) or [3H]-T (final concentration 22 nM), 10 μL unlabeled ligand or solvent, 150 μL incubation buffer, and 100 μL of cytosolic fractions from three preparations of pooled testes or ovaries. Unlabeled ligands E2, Tam (tamoxifen), BPA, T (testosterone), and MT (methyl testosterone) (Sigma, Deisenhofen, Germany) were diluted in 99.8% ethanol and added at final concentrations ranging from 10−9 to 10−3 M. Separation of receptor-bound and free ligands was accomplished by using dextran-coated activated charcoal solution [prepared by adding 3.75 g activated charcoal Norit A (4- to 7- μm particle size) (Serva, Heidelberg, Germany) and 0.375 g dextran T70 (Roth, Karlsruhe, Germany)] to 20 mM Tris–HCl buffer, pH 7.4. Three hundred microliters of charcoal solution were added to each sample, vortexed, incubated for 5 min, and centrifuged for 15 min at 3,500 × g and 4°C. Free ligands were adsorbed by activated charcoal and sedimented. Amounts of [3H]-E2 or [3H]-T bound to binding sites were determined by measuring the radioactivity of 400 μL supernatant in 3 mL scintillation cocktail (Ultima Gold TM;Packard BioScience, Groningen, the Netherlands), using a liquid scintillation counter (Tri Carb 1600; Canberra-Packard, Rodgau-Jugesheim, Germany). Samples were counted for 2 min, and measurements were corrected for background radiation.
The computer programs StatEasy (Wissenschaftliche Auswertungen, Hamburg, Germany) and Prism, version 4.02 (GraphPad Software, San Diego, CA, USA) were used for all statistical analyses. The chi-squared test was applied for mortality data, the t-test was used for the comparison of egg production between identical nominal treatment groups at different temperatures, and the Weir test was employed to detect imposex intensities. Analysis of covariance (ANCOVA) was used to ascertain cumulative egg and clutch numbers, and one-way analysis of variance (ANOVA) with the Student-Newman-Keuls post-hoc test was used to determine egg and clutch numbers per female and to make a comparison of genital organ lengths. The significance level for all tests was set at p < 0.05 when compared to solvent control. BPA half-lives in the exposure tanks were calculated using a one-phase exponential decay model, IC50 (median inhibitory concentration) values with a homologous competitive binding curve for one class of binding sites, and EC10 and EC50 values with a Weibull distribution (Prism 4.02).