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-Ethynylestradiol
Agriculture and Agri-Food Canada, Research Branch, 1391 Sandford Street, London, ON, Canada N5V 4T3
* Corresponding author (toppe{at}em.agr.ca)
Received for publication October 16, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ethynylestradiol in agricultural soil was established in laboratory microcosm studies. The hormone was rapidly dissipated in loam, sandy loam, and silt loam soils under a range of moisture and temperature conditions. Dissipation of 17-ethynylestradiol correlated closely with removal of total estrogenicity determined with a recombinant yeast bioassay, indicating that extractable estrogenic transformation products did not accumulate. The stability of 17-ethynylestradiol in sterile soil, decreased removal in the absence of oxygen, and the response of dissipation kinetics to variation in temperature and moisture suggested that the removal was microbially mediated. We conclude that 17-ethynylestradiol is rapidly dissipated in agricultural soils under a range of conditions typical of a temperate growing season.
Abbreviations: HPLC, high performance liquid chromatography • YES, yeast estrogenicity screen assay
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The synthetic analogue of 17ß-estradiol, 17-ethynylestradiol, is a potent xenoestrogen and is a component of oral contraceptives (Arcand-Hoy et al., 1998). Excreted 17-ethynylestradiol is incompletely degraded in sewage treatment plants, and contributes to the feminization of male fish in rivers receiving sewage outflow (Desbrow et al., 1998; Routledge et al., 1998). Agricultural land could potentially receive 17-ethynylestradiol through the use of sewage biosolids as fertilizer, a common farming practice in proximity to cities (Ternes et al., 1999). Given the paucity of information about the environmental fate of this molecule, we investigated the persistence and pathways of 17-ethynylestradiol dissipation in agricultural soils.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Ethynylestradiol (Fig. 1)
was purchased from the Sigma Chemical Co. (St. Louis, MO). This molecule has the following properties (Lai et al., 2000): molecular weight (g mol-1) = 296.4; octanol–water coefficient (log P) = 3.67; vapor pressure (Pa at 25°C) = 8 x 10-7; and water solubility (mg L-1) = 4.8.
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Soil microcosms were prepared, incubated, and sampled exactly as previously described (Colucci et al., 2001; Topp and Starratt, 2000). In experiments examining the effect of soil moisture content on hormone dissipation, a range of moisture values from 5% to field capacity was established at the start of the 30°C incubation in the above-mentioned soils. The effect of initial hormone concentration (ranging from 0.1 to 10 mg kg-1) and incubation temperature (4, 10, 20, and 30°C) on 17-ethynylestradiol dissipation was determined in the loam soil.
The persistence of 17-ethynylestradiol in anaerobic soils was evaluated in a series of 35-mL serum flasks sealed with butyl rubber stoppers. Each flask contained 5 g of soil supplemented with 1 mg kg-1 of 17-ethynylestradiol. The flasks were made anaerobic by repeatedly evacuating the headspace on a vacuum manifold and backfilling with nitrogen gas through a 22G hypodermic needle. Flasks were periodically sacrificed for extraction and analysis of residual 17-ethynylestradiol.
Sterile soil was prepared by autoclaving twice (45 min at 120°C), the second time following a 24-h room temperature incubation.
Analytical Methods
Preparation of soil extracts, and analytical conditions for high performance liquid chromatography (HPLC) and yeast estrogenicity screen (YES) bioassay of estrogenicity were exactly as described by Colucci et al. (2001) unless otherwise indicated. Preliminary experiments established that the efficiency of 17-ethynylestradiol extraction following addition to the soils used in this study was 80.5 ± 3.3% (n = 5). The HPLC retention time for 17-ethynylestradiol was 12.5 min with a 47:53 solvent blend of acetonitrile and water delivered at 1 mL min-1. The lower limit of detection for 17-ethynylestradiol in extracts prepared from the soils used in this study (100-µL sample injection) was 5 ng. Taking into account our extraction methods, this corresponded to a method detection limit of 50 µg 17-ethynylestradiol kg-1 soil.
Calculations
The dissipation, or decrease in extractable concentration, of 17-ethynylestradiol was determined by HPLC with ultraviolet detection analysis of soil extracts.
Rate constants (expressed throughout as day-1) for dissipation (kD) and loss of total estrogenicity (kE) represent initial reaction rates using data that conformed to first-order kinetics. The coefficient of determination (r2) of linear regressions of the common log of substrate removed over time was used to determine the region of linearity from which the initial rate constants were calculated.
All treatments were in triplicate, and data in figures are expressed as mean ± standard deviation. Statistically significant differences, considered to be at the P < 0.05 probability level, were established by subjecting data to a one-way analysis of variance (ANOVA) test.
Since soil moisture contents were held constant during all incubations, hormone concentrations are expressed on a moist soil basis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Ethynylestradiol was rapidly dissipated in a loam soil incubated at 30°C at initial concentrations ranging from 10 to 0.1 mg kg-1 (Table 1). At 17-ethynylestradiol concentrations of 1 or 10 mg kg-1, the decline in 17-ethynylestradiol concentration was closely accompanied by a decline in total estrogenicity as determined by the YES estrogenicity bioassay (Fig. 2)
. At the end of the 44-d incubation, residual hormone concentration and estrogenicity were below the detection limits for the corresponding analysis. The hormone was completely stable in autoclaved loam soil, similarly incubated (data not shown).
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-ethynylestradiol was determined for the three soils (Table 2). 17-Ethynylestradiol was most persistent in the drier soils. Both the kinetics of hormone dissipation and loss of estrogenicity approximated first order, except in the drier soils, where the coefficients of variation were much lower than 1. In all cases, loss of extractable 17-ethynylestradiol was accompanied by the removal of YES assay-detectable estrogenicity. For all three soils, after 22 d of incubation, removal of hormone and estrogenicity to below detection limits was accomplished generally under conditions of increased moisture.
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-ethynylestradiol was removed much more slowly in the loam soil incubated under an atmosphere of nitrogen than under an atmosphere of air (Fig. 3)
. In sterilized soil, the hormone was stable and persistent during the 14-d incubation period.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ethynylestradiol was rapidly dissipated in agricultural soils ranging widely in texture and properties. The molecule remained stable in sterile soil, and the variation in dissipation rate with temperature, moisture, and oxygen availability was consistent with microbially mediated transformation. We attempted to fit the variable temperature data to the Arrhenius equation, but this was unsatisfactory, probably because of the very high reaction rates. Hormone removal in aerobic nonsterile soil was initiated without a lag phase, and the dissipation kinetics were generally first order, indicating that microbial transformation did not require an adaptive phase, and that there was no proliferation of the biodegrading organisms during the experiments. The close relationship between the removal of 17-ethynylestradiol and the decline in YES-detectable estrogenicity indicates that any product(s) of microbial transformation did not contribute to further estrogenicity.
In the absence of any detectable transformation products we have no direct evidence for the biodegradation pathway. However, oxygen is probably required, given the markedly longer persistence in soil incubated under nitrogen, and slower dissipation in water-saturated fine-textured soils. Degradation pathways of aromatic xenobiotics in aerated environments commonly include ring hydroxylation reactions, with oxygen as a reagent (Topp et al., 1997). 17-Ethynylestradiol metabolism in humans is initiated by hydroxylation, generally at position 2, yielding 2-hydroxy-ethynylestradiol (Arcand-Hoy et al., 1998). It is reasonable to speculate that this compound is transformed by microbially expressed hydroxylases in aerated soils. The dihydroxy product would be very labile to subsequent condensation reactions, resulting in formation of non-extractable residues (Bollag et al., 1998). Taken together, these results suggest that the hormone will be more stable in cold wet soils, and in oxygen-deprived receiving waters or sediments.
In a companion study, we found that 17ß-estradiol degradation occurred with kinetics somewhat faster, but generally comparable with those obtained here for 17-ethynylestradiol degradation (Colucci et al., 2001). A comparison of ethynylestradiol and 17ß-estradiol dissipation over the range of conditions employed in these studies shows that the latter was removed generally two to seven times more rapidly than the former under similar conditions. The only exception was when the soils were adjusted to their field moisture capacities, when both compounds were removed at comparably slow rates. Under these circumstances, the slow diffusion of oxygen presumably limited the rate of transformation of either hormone. These results are in agreement with the longer persistence of 17-ethynylestradiol than 17ß-estradiol during sewage treatment (Ternes et al., 1999). We have readily obtained enrichment cultures from our soils that use 17ß-estradiol as the sole carbon source, whereas attempts to obtain bacterial enrichment cultures from the same soils that grow at the expense of 17-ethynylestradiol have failed (data not shown). The estradiol enrichment cultures mineralize [4-14C]-17ß-estradiol, but do not transform 17-ethynylestradiol. Thus, although these hormones are very rapidly removed in soil, the initial attack on the molecules is probably initiated differently.
The likelyhood of 17-ethynylestradiol reaching agricultural land in sewage biosolids is uncertain, in particular because the affinity of hormones to the solid phase and likely partioning behavior during sewage treatment need to be clarified (Daughton and Ternes, 1999; Shore et al., 1993; Alcock et al., 1999; Desbrow et al., 1998). This study is to our knowledge the first reporting mechanisms of ethynylestradiol dissipation in soil, and suggests that this xenoestrogen will be readily biodegraded should it reach agricultural soils.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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