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TECHNICAL REPORT
Organic Compounds in the Environment

Persistence of Estrogenic Hormones in Agricultural Soils

I. 17ß-Estradiol and Estrone

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.

	ABSTRACT

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The persistence and pathways of dissipation of 17ß-estradiol and estrone in soil were established in laboratory microcosm incubations. [4-¹⁴C]-17ß-Estradiol dissipation and mineralization rates were determined over a range of temperatures and moistures, and this compound was rapidly removed in soil conditions typical of a temperate growing season. 17ß-Estradiol was oxidized to estrone in both autoclaved and nonsterile loam, silt loam, and sandy loam soils, suggesting an abiological transformation. In contrast, estrone was stable in autoclaved soil, suggesting that its removal was microbially mediated. Both [4-¹⁴C]-17ß-estradiol and [4-¹⁴C]-estrone formed non-extractable residues, and soil-bound residues were only slowly mineralized, suggesting that their bioavailability was low. Determination of total estrogenicity in soil extracts by means of a recombinant yeast assay indicated that there were no other estrogenic compounds produced during 17ß-estradiol dissipation, and that total estrogenicity was rapidly dissipated below the detection limit. We suggest that environmental studies evaluating the movement and persistence of estrogenic hormones from animal wastes should include estrone in their analyses.

Abbreviations: EDC, endocrine disrupting chemical • HPLC, high performance liquid chromatography • LSC, liquid scintillation counting • YES, yeast estrogenicity screen assay

	INTRODUCTION

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THERE is growing concern about the apparently increasing incidence of reproductive disorders and abnormal development in wildlife and reduced fertility in human males, problems that may be caused by so-called endocrine disrupting chemicals (EDCs) released anthropogenically into the environment (Ashby et al., 1997; Sonnenschein and Soto, 1998). Known or suspected man-made endocrine disruptors include industrial chemicals such as dioxins, polychlorinated biphenyls (PCBs), and nonylphenols, and a number of now-banned pesticides such as DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] (Safe and Gaido, 1998; Kavlock et al., 1996). Endocrine disrupting chemicals possess the ability to alter or disrupt endocrine system function by mimicking, antagonizing, or interfering with the biosynthesis or biodegradation of endogenous hormones (Sonnenschein and Soto, 1998). Perhaps the clearest evidence of endocrine disruption in wildlife are reproductive abnormalities observed in fish or reptiles exposed to effluents from sewage treatment plants or pulp and paper mills, and possibly agricultural runoff (Jobling et al., 1998; Harries et al., 1997, Munkittrick et al., 1997; Guillette et al., 1994). Agriculture could potentially be a source of environmental EDCs through the use of certain pesticides, land application of sewage biosolids, or land application of animal wastes containing elevated levels of hormones such as 17ß-estradiol and estrone (Fig. 1) excreted by poultry or livestock (Peterson et al., 2000; Short and Colborn, 1999; Ternes et al., 1999b; Nichols et al., 1997; Shore et al., 1995; Giger et al., 1984). 17ß-Estradiol and estrone are bioactive at remarkably low environmental concentrations. For example, the induction of vitellogenin production in male fish occurs at concentrations as low as 1 ng L^-1 (Purdom et al., 1994). Furthermore, mixtures of estrogenic compounds may act in combination to produce an enhanced response, complicating the risk assessment of mixed effluents such as agricultural runoff (Daughton and Ternes, 1999; Sumpter and Jobling, 1995). A number of recent studies have revealed movement of 17ß-estradiol from manured land into surface and ground water in concentrations that could affect wildlife (Peterson et al., 2000; Bushée et al., 1998; Nichols et al., 1997).

View larger version (14K): [in this window] [in a new window]   Fig. 1. Structures of the estrogenic hormones 17ß-estradiol and estrone. [4-14C]-17ß-Estradiol and [4-14C]-estrone were labeled in the indicated positions.

	MATERIALS AND METHODS

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Chemicals
17ß-Estradiol and estrone (Fig. 1 and Table 1) were purchased from the Sigma Chemical Co. (St. Louis, MO). [4-¹⁴C]-17ß-Estradiol (1.92 TBq mol^-1) and [4-¹⁴C]-estrone (2.07 TBq mol^-1) were purchased from NEN Life Sciences Products (Boston, MA). All radiolabeled hormones had a radioactive purity greater than 97%.

View this table: [in this window] [in a new window]   Table 1. Physical and chemical properties of 17ß-estradiol and estrone.

Samples for hormone residue analyses were taken periodically by opening the mason jars and removing 5 g moist soil by means of a sterile spatula. Soil samples were extracted immediately, or stored at -20°C in glass scintillation vials.

Sterile soil was prepared by autoclaving twice (45 min at 120°C), the second time following a 24-h room temperature incubation.

Analytical Methods
Radioactivity was measured with a Model LS 5801 liquid scintillation counter (Beckman Instruments, Irvine, CA). Each sample received 10 mL of UniverSol scintillation cocktail (ICN, Cosa Mesa, CA). Quenching was corrected using an external standard.

Parent compounds and transformation products were analyzed by reverse phase high performance liquid chromatography (HPLC) with ultraviolet (Varian [Palo Alto, CA] 9050 variable wavelength UV-VIS detector) and radioactivity (EG&G Berthold [Bad Wildbad, Germany] LB509 radioflow detector) detection. Instrument operating conditions were as follows: column, 25.0 cm x 4.6 mm (10-µm Partisil 10 ODS-3 packing); mobile phase, acetonitrile and water (40:60); UV detector wavelength, 220 nm. With solvent delivered at 1 mL min^-1, 17ß-estradiol had a retention time of 18 min, and estrone 22 min. The lower limit of detection for both estrone and 17ß-estradiol in extracts prepared from the soils used in this study (100 µL sample injection) was 5 to 7.5 ng. Taking into account our extraction methods, this corresponded to a method detection limit of 50 to 75 µg 17ß-estradiol or estrone kg^-1 soil.

Mass spectra were determined by electron impact on a Finnigan-MAT (Bremen, Germany) 8230 mass spectrometer, at an ionizing voltage of 70 eV. Metabolites were isolated and purified in preparation for mass spectral analysis by fractionating soil extracts using HPLC, evaporating under a stream of nitrogen, and taking up the residue in methanol.

The estrogenicity of soil extracts was determined using the yeast estrogenicity screen (YES) assay as described by Routledge and Sumpter (1996), except that the chromogen detection wavelengths were 550 nm and 630 nm. The gene encoding the human estrogen receptor (hER) protein has been cloned into a strain of Saccharomyces cerevisiae. The strain also contains a plasmid carrying the gene encoding the enzyme ß-galactosidase (lacZ) under the transcriptional control of human estrogen–responsive sequences (ERE). Molecules that enter the yeast and bind with hER form a ligand, which interacts with ERE, activating transcription of lacZ, and the expression of ß-galactosidase. Hydrolysis of chlorophenol red-ß-D-galactopyranoside (CPRG) by ß-galactosidase forms a red chromogen, the concentration of which is linearly related to the estrogenicity of the sample, expressed here as 17ß-estradiol equivalents. The method detection limit for the YES assay determined with standards prepared in extracts from the soils used in this study was 1 µg estradiol kg^-1 soil.

Unless otherwise indicated, samples for various analyses were prepared by extracting 5-g portions of soil either twice with ethyl acetate (1:1) or once with ethyl acetate (1:1) followed by acetone (1:1; 1 h with agitation at 30°C). Samples were taken to dryness in a glass tube under a stream of nitrogen, and taken up in acetonitrile for HPLC or LSC analysis, or absolute ethanol for recombinant yeast estrogenicity screen (YES) assay analysis. Preliminary experiments established that the efficiency of estradiol extraction following addition to the soils used in this study was 98.9 ± 10.4% (n = 9), and for estrone 72.4 ± 4.4% (n = 6).

Calculations
The dissipation, or decrease in extractable concentration, of hormones was determined by HPLC analysis of soil extracts. The formation of non-extractable residues was calculated from the decrease in extractable radioactivity measured in extracts by LSC. This procedure was judged to be appropriate since mineralization of radiolabel during mass-balance determination experiments was found to be insignificant during incubations of less than one week. Mineralization of ¹⁴C-labeled hormones was determined by measuring liberated ¹⁴CO₂ in NaOH traps by LSC and calculating the percentage of the total radioactivity represented by the accumulated radioactivity. Extractable radioactivity is presented as a percentage of the initial radioactivity added.

Rate constants (expressed throughout as day^-1) for dissipation (k_D) and loss of total estrogenicity (k_E) 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.

	RESULTS

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17ß-Estradiol was rapidly removed in agricultural soils incubated under a range of conditions. At 30°C, following supplementation with 1 mg kg^-1 [4-¹⁴C]-17ß-estradiol, the quantity of extractable radioactivity rapidly decreased (Fig. 2 and Table 3). Following 3 d of incubation, 90.7% of the radioactivity initially applied to the loam soil was non-extractable. In the sandy loam and silt loam soils, the non-extractable radioactivity was 70.3 and 56.0%, respectively. The HPLC with radioactivity detection analysis of the extracts indicated that 17ß-estradiol was removed rapidly in all three soils, with a DT₅₀ (i.e., time to dissipate 50% of the initial concentration) of less than 0.5 d in all cases. Removal of 17ß-estradiol was accompanied by the accumulation of another radiolabeled product, with a retention time identical to that of an estrone standard. This transformation product was isolated from a soil extract, and subjected to solid probe mass spectrometry. The compound had a mass spectrum identical to that of an analytical standard of estrone (molecular formula C₁₈H₂₂O₂), with a molecular ion at m/z 270 (M⁺) and major fragments at m/z 242, 226, 213, 185, 146, and 115 (data not shown). In the loam soil, estrone accumulated transiently and maximally at 6 h, and was undetectable thereafter. In contrast, in the silt loam and sandy loam soils, estrone remained detectable throughout the experiment, and comprised 100% of the extractable radioactivity by the end of the experiment. Throughout the incubation radioactive estradiol and estrone together were sufficient to account for all of the extractable radioactivity. Taking into account that estrone has half the estrogenicity of 17ß-estradiol in the YES assay, removal of total extractable estrogenicity, measured with the YES assay, closely parallelled the decline in total extractable radioactivity. Taken together, these results indicate that there were no other transformation products, estrogenic or otherwise, accompanying the degradation of estradiol and estrone.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Persistence of 1 mg kg-1 [4-14C]-17ß-estradiol in a sandy loam soil (triangle), a silt loam soil (square), and a loam soil (circle) incubated at 30°C. Soils were adjusted to a moisture content of 13%. The soils were nonsterile (left panels) or autoclaved (right panels). Top panels: total extractable radioactivity expressed as a percentage of that initially applied. Middle panels: distribution of radioactivity in 17ß-estradiol (open symbols) and estrone (closed symbols) determined by high performance liquid chromatography (HPLC) with radioactivity detection, and expressed as a percentage of the total extractable radioactivity in that sample. Bottom panels: total estrogenic activity in soil extracts measured with the yeast estrogenicity screen (YES) bioassay, and expressed as 17ß-estradiol equivalents.

[4-¹⁴C]-17ß-Estradiol was mineralized only very slowly by the three soils incubated at 30°C (Table 3). By the end of a 3-mo incubation, 11.5 to 17.1% of the initially applied estradiol-¹⁴C was recovered as ¹⁴CO₂.

There were significant differences in the rates of [4-¹⁴C]-estrone removal in the three soils, with maximal rates noted for the silt loam soil followed by the loam and sandy loam soils (Table 3). There was no significant difference in estrogenicity dissipation between the loam and silt loam soils, but estrogenicity was removed more slowly in the sandy loam soil. In all three soils, the formation of non-extractable residues was observed.

The initial rates of 17ß-estradiol dissipation in the loam soil did not vary greatly with temperature, except at 4°C where the rate was significantly lower compared with the rates observed at higher temperatures (Table 4). Temperature had likewise no significant effect on the rate of loss of total estrogenicity. Following 32 d of incubation, both hormone concentrations and estrogenicity were below the detection limits of the corresponding analytical procedures. Mineralization of [4-¹⁴C]-17ß-estradiol was more responsive to temperature, with optimal and comparable ¹⁴CO₂ yields at 30 and 37°C (Fig. 3 and Table 4).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effect of temperature on mineralization of 1 mg kg-1 [4-14C]-17ß-estradiol in a loam soil adjusted to a moisture content of 13%. The soil was incubated at 4°C (closed circles), 10°C (open circles), 19°C (closed inverted triangles), 30°C (open inverted triangles), or 37°C (closed squares).

	DISCUSSION

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Assessing the relative significance of potential rate-controlling parameters such as soil type, temperature, and moisture on the various measures of hormone degradation was complicated by the very rapid reaction rates. Nevertheless, some conclusions are clear. The dissipation pathway, conversion of estradiol to estrone, and subsequent formation of non-extractable residues was consistent in all soils under all incubation conditions. A comparison of three soil types, ranging in texture, in pH from 7.4 to 5.8, and in organic matter content from 3.2 to 0.8%, does not reveal any consistent effect of these soil properties on dissipation rate. Hormone removal, measured either chemically or by means of the YES bioassay, was relatively insensitive to temperature, other than at 4°C. In contrast, mineralization of [4-¹⁴C]-17ß-estradiol did respond more dramatically to temperature; however, the extent of mineralization after ca. 2 mo was minimal. Finally, dissipation was slower when soils were air-dried or adjusted to field moisture capacity, consistent with limitation of diffusion of substrates in the aqueous phase, or oxygen through the gas phase, respectively. Overall, other than when soil is extremely dry or very cold, these hormones would be expected to dissipate with DT₅₀ values (i.e., time to dissipate 50% of the initial concentration) in the order of hours or a few days.

We attempted to fit the variable temperature dissipation data to the Arrhenius equation, but this was unsatisfactory because of the generally insignificant effect of temperature on dissipation through the range tested (Table 4).

In nonsterile soil, [4-¹⁴C]-17ß-estradiol and [4-¹⁴C]-estrone readily formed non-extractable residues, whereas in sterile soils the radioactivity remained largely extractable during incubations lasting several days. This suggests that microbially mediated transformation of estrone is required for the formation of non-extractable residues, although the profound changes in soil composition caused by autoclaving, which could affect sorption or sequestration, cannot be discounted. The ready formation of non-extractable residues, absence of any additional radiolabeled transformation products in extracts, and very slow mineralization of [4-¹⁴C]-17ß-estradiol suggest that the estrone transformation product(s) were highly reactive with the soil, and that the non-extractable residues were relatively nonbioavailable. In contrast, for example, the rates of mineralization of ¹⁴C-labeled rye (Secale cereale L.) residues in these soils in comparable experiments were much higher (Topp et al., 1998). The k_dissipation for 17ß-estradiol was higher than that for estrone, consistent with the accumulation of this latter compound as a transient transformation product (Table 3).

Estrone removal and 17ß-estradiol mineralization occurred without a lag phase, in contrast to the behavior of many xenobiotics, which are detectably metabolized only after repeated application (Racke and Coats, 1990). Therefore, in these soils, microorganisms could transform the hormones without need for prior adaptation. 17ß-Estradiol and other structurally related steroidal hormones are involved in the developmental regulation of many invertebrates and all vertebrates, and can therefore be expected to be environmentally ubiquitous (Cheek et al., 1998). The generally first-order kinetics of the dissipation processes measured in this study suggest that proliferation of biodegradative organisms did not occur during the incubations.

By means of the YES assay, total estrogenicity in soil extracts was followed throughout the incubations. The close relationship between estrone removal and loss of estrogenicity indicated that there were no other extractable estrogenic metabolites produced during the dissipation of 17ß-estradiol. In our hands, on a molar basis, estrone had half the estrogenicity of 17ß-estradiol in the YES assay, and therefore this transformation would significantly reduce estrogenicity in estradiol-contaminated environmental samples (data not shown). The lability of 17ß-estradiol in soils is consistent with what is observed during sewage treatment, where rapid conversion to estrone occurs, and the ratio of estrone to 17ß-estradiol in sewage outflows is high, suggesting that this transformation is strongly favored (Ternes et al., 1999a, b; Desbrow et al., 1998).

Estrogenic hormones are generally excreted in biologically inactive conjugated forms (Desbrow et al., 1998). These hormone conjugates are readily hydrolyzed back to the estrogenic hormone by bacteria producing ß-glucuronidase or sulfatase enzymes (Daughton and Ternes, 1999; Desbrow et al., 1998; Ternes et al., 1999a). Conjugates can reasonably be expected to be readily and rapidly hydrolyzed in the environment (Alcock et al., 1999; Desbrow et al., 1998).

Recently, 17ß-estradiol has been detected in ground and surface waters adjacent to land manured with livestock or poultry waste (Peterson et al., 2000; Finlay-Moore et al., 2000; Bushée et al., 1998; Nichols et al., 1997). Our study has established that 17ß-estradiol and estrone are labile in soil, and elucidated dissipation pathways, but further work is warranted to clarify the fate of these compounds in the field. First, for practical analytical reasons, the hormone concentrations we used were much higher than what could reasonably be expected in situ. Second, the mass balance and distribution of parent compounds and transformation products in the soil were established using rigorous solvent extraction methods. The proportion of non-extractable residues obtained is operationally defined, and varies according to the rigor of the extraction procedure. In the field, the solvent is water, and the proportion of non-extractable residues is therefore likely to be higher than that obtained by extraction with organic solvents under optimized conditions (Alexander, 2000). Overall, it will be important to establish soil interactions, leaching potential via matrix and preferential flow, and dissipation kinetics with much lower hormone concentrations. The persistence of these chemicals once they reach surface or ground water needs to be established.

Reported field studies have quantified 17ß-estradiol with commercially available enzyme immunoassays, which do not detect estrone with any efficiency (Peterson et al., 2000; Nichols et al., 1997; Bushée et al., 1998). Based on our results, it would seem important to include estrone in environmental surveys of estrogen contamination of ground or surface water.

In conclusion, 17ß-estradiol and estrone were readily biodegradable in soils under a range of temperature and moisture conditions and we predict that they would be rapidly dissipated in aerated agricultural soils following application of manures during a temperate growing season. These results further affirm that manure application methods which maximize contact with soil and minimize the likelyhood of surface or bypass flow would best protect ground and surface waters from contamination with chemicals of concern.

	ACKNOWLEDGMENTS

 
This work was funded by the Canadian Toxic Substances Research Initiative. Nicolas Adenot provided excellent technical assistance. We thank Ralph Chapman for advice on extraction procedures.

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