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a Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
b Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V 4T3
* Corresponding author (toppe{at}agr.gc.ca)
Received for publication August 25, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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-androstan-3,17-dione and 1,4-androstadiene-3,17-dione, were also detected. Experiments with sterilized soil and sterilized swine manure slurry suggested that the transformation of 14C-labeled hormonal parent compounds was mainly caused by microorganisms in manure slurry, while mineralization of the hormones to 14CO2 required viable soil microorganisms. Organic amendments transiently inhibited the mineralization of [4–14C]-testosterone, perhaps by inhibiting soil microorganisms, or by enhancing sorption and reducing the bioavailability of testosterone or transformation products. Overall, organic amendments influenced the pathways and kinetics of testosterone and estradiol dissipation, but did not increase their persistence.
Abbreviations: HPLC, high performance liquid chromatography • LSC, liquid scintillation counting • RD, radioactivity detection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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The major pathways of environmental exposure to steroidal hormones are likely effluent from sewage treatment plants, and the agricultural use of municipal sewage sludge (biosolids) and animal manures as fertilizer. A recent survey by Lorenzen et al. (2004) of estrogenic and androgenic activity in municipal biosolids, dairy, beef, swine, and poultry waste using hormone receptor binding assays detected a range of hormonal activities in these materials, with the highest levels of estrogenic activity in manure slurry from finishing pigs (5965 ng estradiol equivalents/g dry wt.) and the highest levels of androgenic activity in manure from pregnant dairy cows (1737 ng testosterone equivalents/g dry wt). It is estimated that farm animals in the United States excrete 49 Mg of estrogenic and 4.4 Mg of androgenic hormones per year, and another 1 Mg of androgenic hormones are administered as growth-promoting agents (reviewed in Lange et al., 2002). Finlay-Moore et al. (2000) measured soil concentrations of up to 675 ng/kg 17ß-estradiol and 260 ng/kg testosterone in fields amended with broiler litter. In the same study hormone concentrations in surface runoff reached 50 to 2300 ng/g 17ß-estradiol and 10 to 1830 ng/L testosterone. 17ß-estradiol has been detected in ground water adjacent to fields amended with poultry litter and cattle manure, in concentrations of 6 to 66 ng/g (Peterson et al., 2000).
Given the continued intensification of livestock and poultry production and the widespread urbanization in areas proximal to areas of crop production, the pressure to accommodate more animal or human waste on agricultural land will likely increase. We and others have therefore investigated the fate of steroidal hormones in agricultural soils. In the laboratory, both testosterone and 17ß-estradiol are rapidly dissipated with half-lives typically ranging from a few hours to a few days in a range of soils incubated under a range of temperature and moisture conditions (Lee et al., 2003; Casey et al., 2003, 2004; Colucci et al., 2001; Colucci and Topp, 2002; Das et al., 2004; Hanselman et al., 2003; Lorenzen et al., 2005). In the present study, we investigated the effect of manure or biosolids, the materials that can carry the hormones to agricultural soil, on hormone dissipation in soils. These organic matrices, rich in nutrients, organic matter, and microorganisms, will profoundly change soil conditions following application, and therefore could influence the persistence and pathways of hormone dissipation, perhaps altering the risk of contamination of adjacent water to hormones.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Soils and Organic Amendments
Loam (pH of 7.4; 3.2% organic matter; 40% sand, 45% silt, 15% clay), sandy loam (pH of 5.8; 0.8% organic matter; >90% sand), and silt loam (pH of 6.7; 2.9% organic matter; 32% sand, 52% silt, 16% clay) soils used in this study have previously been described in Colucci et al. (2001). Soils were sieved to 2-mm maximum particle size and air-dried to a 7% moisture content immediately before experiments. Sterile soil was prepared by autoclaving twice for 45 min at 120°C, the second time following a 24-h room temperature incubation. Experiments were performed using the loam soil, unless otherwise stated.
Manures were obtained from the open-air lagoons of four different commercial swine-producing farms that handle their wastes as a slurry. The sewage sludge used in this study was obtained from a municipal wastewater treatment plant serving about 60000 residents and having negligible industrial input in the city of London, Ontario. The chemical properties of manures and sewage sludge (biosolid) are listed in Table 1. Manure and biosolid properties were determined using standard methods (A&L Canada Laboratories East, London, ON, Canada). For some experiments organic amendments were sterilized by autoclaving twice for 20 min at 120°C. Soils, manures, and biosolids were stored at 4°C before experiments.
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Parent compounds and transformation products were analyzed by reverse phase high performance liquid chromatography with UV and radioactivity detection (Model LB509 radioflow detector; Berthold, Bad Wildbad, Germany). Instrument operating conditions for analysis of 17ß-estradiol were as follows: column, 25.0 cm x 4.6 mm (10-µm Partisil 10 ODS-3 packing; Whatman, Maidstone, UK); mobile phase, acetonitrile and water (40:60); UV detector wavelength, 220 nm. With solvent delivered at 1 mL/min retention times were 17.6 min for 17ß-estradiol and 21.5 min for estrone. The analysis of testosterone and transformation products was done using the same procedures, except the mobile phase consisted of acetonitrile and water (37:63) delivered at 1.15 mL/min. Under these conditions, testosterone (29 min), 4-androstene-3,17-dione (33 min), 5-androstan-3,17-dione (55 min), and 1,4-androstadiene-3,17-dione (23 min) had the indicated retention times. The identity of transformation products was confirmed by HPLC–mass spectrometer detection as described in Lorenzen et al. (2005).
Methods for hormone receptor gene transcription bioassay were exactly as in Lorenzen et al. (2005).
Soil Microcosms
The rate of testosterone and 17ß-estradiol mineralization and dissipation pathway in soils with or without organic amendment were investigated using microcosm studies as described in Lorenzen et al. (2005). Microcosms consisted of 50-g portions of soil (moist weight) dispensed in baby-food jars incubated in a sealable 1-L Mason jar containing a vial with 10 mL of water to maintain moisture and a vial with 5 mL of 1 M NaOH solution for trapping 14CO2. Soils were supplemented with 14C-labeled testosterone or 17ß-estradiol as follows. Ethanolic stock solutions of hormones were added by pipette to 1-g portions of pulverized air-dried soil. The solvent was allowed to evaporate and the soil was thoroughly incorporated into the 50 g of moist soil in baby jars. The initial hormone concentration were 81.0 µg [4–14C]-testosterone or 81.7 µg [4–14C]-17ß-estradiol per kg soil (moist weight), corresponding to a radioactivity of 30000 dpm per gram soil. In addition to labeled compounds the soil received 1 mg cold testosterone or 17ß-estradiol per kg soil. Finally, 5-mL amendments (swine manure slurry, biosolids, or water) were added per 50 g of soil in microcosms and thoroughly incorporated. Soil moisture contents were normalized to 15% in all treatments by addition of distilled water. Microcosms were incubated in the dark at 30°C, unless otherwise indicated. Mason jars were opened regularly (i.e., for soil sampling) and aerobic conditions were maintained in the test system.
All treatments were prepared in triplicate and data are reported as mean ± standard deviation. Soil concentrations are expressed on a moist soil basis, as moisture content was held constant during incubations. Extractable radioactivity is calculated as the percentage of the initially added radioactivity. Mineralization is the accumulated radioactivity calculated as the percentage of initially added radioactivity trapped as 14CO2 in the NaOH solution.
In this study, data reported as being significantly different were considered to be so at the 0.05 probability level. Differences were established by subjecting data to a one-way ANOVA test.
Sampling and Soil Extraction
Soil samples (5 ± 0.05 g) were taken from each jar immediately after starting the experiment (t = 0) and after 6, 24, 48, and 144 h, unless otherwise indicated. Hormones were extracted from the soil with 2 x 10 mL ethyl acetate and once with 10 mL of acetone, as described in Lorenzen et al. (2005). For each extraction samples were vigorously shaken automatically for 10 min on a wrist action shaker (Burrell Corporation, Pittsburgh, PA). The sample was then centrifuged (Model GLC-1, 900 rpm [164 x g]; Sorvall, Asheville, NC) for 10 min and the supernatant transferred to a clean glass vial. The pooled supernatants were reduced to dryness under nitrogen in a 37°C water bath and redissolved in 500 µL of ethanol. A quarter of the final extract was used for LSC analysis of total extractable radioactivity, while the remaining three-quarters were analyzed for degradation products using HPLC–radioactivity detection (RD). For measuring mineralization as 14CO2 trapped in the NaOH solution, the complete vial containing 5 mL of NaOH was replaced and 10 mL of LSC cocktail added before LSC analysis.
Recovery experiments established that the efficiencies of testosterone extraction from unamended and manure-amended loam soil were 84 ± 2 and 89 ± 4%, respectively, and the extractabilities of 4-androstene-3,17-dione were 84 ± 9 and 85 ± 6%, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Soils
The effect of swine manure slurry amendment on testosterone dissipation was further evaluated in three agricultural soils differing in texture and chemical properties. In all three soils, the addition of 10% (v/w) manure transiently but significantly inhibited the mineralization of [14C]-testosterone in comparison with unmanured soils (Fig. 2)
. The inhibition was particularly marked in the silt loam soil. Testosterone mineralization activity generally recovered to comparable levels in all treatments, and by the end of 6 d of incubation there was no significant difference in the total amount of 14CO2 accumulated, with the exception of the sandy loam soil. In this case, 47% of the 14C was recovered as 14CO2 in the manured treatment, in comparison with only 36% in the corresponding unmanured control, indicating enhanced mineralization in manure-amended soil. At the same time that swine manure slurry inhibited testosterone mineralization, it enhanced the recovery of extractable radioactive residues (Fig. 2). This was particularly noteworthy for the silt loam soil, where, for example, at 48 h of incubation, fully 80% of the initial radioactivity was recovered in the manured treatment, whereas less than 20% was extractable from the corresponding unmanured control. At the end of 6 d of incubation, less than 5% radioactivity was extractable in any of the treatments, except manure-amended silt loam (6.8% extractable).
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-androstan-3,17-dione were both in excess of 10% of the initial testosterone concentration. Overall, cooler soil temperatures notably increased the persistence of testosterone transformation products in manured soils.
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-androstan-3,17-dione and trace amounts of 1,4-androstadiene-3,17-dione accumulated transiently in unamended soil as well as soil amended with sterile or nonsterile manure (Fig. 6, Panels A1 and A2). The addition of sterilized manure to nonsterile soil had the same effect on extractability and mineralization of 14C as did the nonsterile manure (compare Fig. 6, Panels A1 and B1). However, in contrast to the effect of nonsterile manure, the conversion of testosterone to 4-androstene-3,17-dione was not accelerated by sterile manure (compare Fig. 6, Panels A2 and B2). There was no mineralization of [14C]-testosterone in sterile soil regardless of the addition of nonsterile manure, and 14C residues remained fully extractable during the course of the incubation (Fig. 6, Panel C1). In sterile soil, testosterone was stable until 48 h of incubation, by which time the soil could reasonably be assumed to have become contaminated with airborne bacteria (Fig. 6, Panel C2). When the sterile soil was supplemented with nonsterile manure, however, testosterone was very rapidly converted to 4-androstene-3,17-dione, which following 6 h of incubation represented 80% of the extractable radioactivity (Fig. 6, Panel C2). Thereafter, 1,4-androstadiene-3,17-dione and 5-androstan-3,17-dione accumulated, although to lower concentrations (Fig. 6, Panel C2). Finally, when both the soil and the manure were sterile, there was no dissipation of testosterone whatsoever (Fig. 6, Panels D1 and D2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Microbial Degradation
Several lines of evidence suggest that microorganisms carried in swine manure slurry contribute to the transformation of testosterone in manured soils. Testosterone was rapidly converted to 4-androstene-3,17-dione in manured soil (Fig. 6, Panels A2 and C2). This reaction also occurred in unmanured soil, but much more slowly (Fig. 6, Panel A2). The ability of swine manure slurry to effect this rapid transformation was entirely lost if the material was sterilized by autoclaving before addition to soil (Fig. 6, Panel B2). When swine manure slurry was added to sterilized soil, all three testosterone metabolites accumulated to significant concentrations, much higher than in nonsterile soil (Fig. 6, Panel C2). The same three transformation products were detected in nonmanured soil (Lorenzen et al., 2005). Mineralization of [14C]-testosterone did not occur in sterilized soil. Finally, the addition of manure reduced the rate of mineralization of [14C]-testosterone, and consequently increased the availability of extractable 14C residues over time (Fig. 1). Taken together, these results suggest that microorganisms carried in swine manure slurry are able to convert testosterone to the three steroidal transformation products. This suggestion is substantiated in that the same transformation products are observed when [14C]-testosterone is added directly to swine manure slurry, incubated either with or without aeration (data not shown). The accumulation of steroidal transformation products in sterilized soil receiving nonsterile swine manure slurry, and the total absence of [14C]-testosterone mineralization in sterile soil, indicates that microorganisms in the soil are required to completely degrade the molecule. The slower rate of mineralization in manured soils suggests that the swine manure is inhibitory or toxic to these organisms. Swine manure slurry can have a significant fumigant effect in soil due to, for example, volatile fatty acids (Tenuta et al., 2002).
Bioavailability
Another potential contributing factor to the reduction in mineralization of [14C]-testosterone in the presence of manure could be enhanced sorption and reduced bioavailability due to the additional organic matter added to the soil. The extraction efficiencies for 1,4-androstadiene-3,17-dione and testosterone in the loam soil were comparable and not affected by the presence of manure, suggesting that the bioavailability of the two compounds should be comparable. However, in the field the solvent is water and the extractability of compounds using organic solvents may not reflect bioavailability. In several studies testosterone and 1,4-androstadiene-3,17-dione sorption was shown to correlate with the soil content of organic matter (Lee et al., 2003; Casey et al., 2003, 2004), which would correspond to stronger sorption in organic amended soils. Furthermore, 1,4-androstadiene-3,17-dione sorption was shown to be stronger than testosterone sorption, also when the sorption coefficients were normalized to the organic carbon content of soil, with average log Koc = 3.34 for testosterone and 3.72 for 1,4-androstadiene-3,17-dione (Lee et al., 2003). Therefore, 1,4-androstadiene-3,17-dione, which is rapidly formed when manure is applied, could be less bioavailable than testosterone, contributing to the lower rate of mineralization and dissipation in organic amended soils. Overall, however, in either the presence or the absence of organic amendment, testosterone is rapidly dissipated in soil, steroidal transformation products transiently accumulate, and both parent compound and products are dissipated within a few hours or days over a range of soil conditions (this study; Lorenzen et al., 2005).
17ß-Estradiol
17ß-estradiol was converted to estrone much more rapidly in manured than in unmanured soils, consistent with the analogous conversion of testosterone to 4-androstene-3,17-dione. In contrast to testosterone, however, swine manure slurry enhanced the mineralization of [14C]-17ß-estradiol somewhat. However, the rate of 17ß-estradiol mineralization in the absence of manure was very slow and, in comparison with testosterone mineralization rates, negligible (Colucci et al., 2001). Corresponding to testosterone, differential sterilization experiments indicate that microorganisms carried in manure can convert 17ß-estradiol to estrone, and that mineralization of [14C]-17ß-estradiol requires a viable soil microbial population (Fig. 7).
Manures and Biosolids
The potency of swine manure slurries varied widely with respect to their effect on testosterone transformation (Fig. 3). Manure II was the most inhibitory, and Manure IV the most benign. There was no obvious relationship between potency and manure physical or chemical properties (Table 1). The most noteworthy variable properties are the manure dry matter and corresponding organic matter content, which are markedly higher in Manures I and II. As discussed above, a higher content of organic matter may result in lower bioavailability of testosterone and transformation products due to stronger sorption, causing a reduced rate of mineralization. The pH values of the manures also reflect the relative effect on testosterone dissipation. However, the loam soil and Manure I both have pH of 7.4 and hence it is not likely that the addition of Manure I would change pH in the samples and thereby cause the observed effects on dissipation.
The inhibition of mineralization of [14C]-testosterone by municipal biosolids was concentration dependent. The slower testosterone mineralization in loam soil heavily amended with sewage sludge could be due to oxygen limitation promoted by high biological oxygen demand and restricted gaseous diffusion, or inhibition of microbial activity due to toxicity of the biosolids. In comparable experiments, there was a similar relationship between biosolids concentration and mineralization of [ring-U–14C] 4-nonylphenol in soil (Topp and Starratt, 2000).
Soils and Temperature
The effects of swine manure slurry on testosterone dissipation observed in the loam soil were also qualitatively consistent in the sandy loam and silt loam soil (Fig. 2), except that mineralization was increased by addition of manure in the sandy loam. The level of mineralization in the unamended sandy loam soil was much lower than all other soils. Likewise, in another study, testosterone was more persistent in sediment with high sand and low organic carbon content, compared with a range of other soils (Lee et al., 2003). Hence, manure amendment may enhance testosterone mineralization in carbon-deficient soils where conditions for testosterone degradation are otherwise poor. In the silt loam, however, the initial inhibition of testosterone mineralization was more pronounced than observed in the loam soil. The rates of mineralization in the unamended soils were comparable, thus the microbial population in the silt loam soil seems to be able to degrade testosterone. The addition of manure may therefore somehow inhibit microorganisms or result in very strong sorption due to the combination of elevated levels of organic carbon and higher surface area in the silt loam, which were both shown to correlate to testosterone sorption (Casey et al., 2004). However, strong sorption of parent compound and transformation products was shown not to hinder degradation, as the high rate of degradation can only be reached if compounds are either degraded while sorbed or rapidly desorbed from the solid phase and then degraded in the aqueous phase (Das et al., 2004).
In most experiments the microcosms were incubated at 30°C, but the effects of organic amendment on testosterone mineralization and extractability were qualitatively consistent at lower temperatures (Fig. 5). Not surprisingly, the rates of degradation were slower at reduced temperatures, presumably due to reduced microbial activity. However, even at 4°C testosterone was completely dissipated within 7 d and less than 10% of the added radioactivity was extractable after 12 d.
Bound Residues and Degradation Products
The biphasic kinetics of testosterone mineralization are characteristic of readily metabolizable substrates that are mineralized for energy and assimilated biosynthetically for growth. The fact that little of the remaining soil radioactivity was extractable is in agreement with this hypothesis. We did not, however, further characterize the nature of the non-extractable residues, which could variously be in the form of bound parent compound, transformation products, or, as we suggest, microbial biomass. The absence of significant extractable testosterone residues evident by HPLC–RD indicates that significant amounts of this residue do not in fact accumulate, or if they do they are effectively bound and not bioavailable.
The hormone degradation products detected in this study generally have lower activities than the parent compound. The major testosterone transformation product detected, 4-androstene-3,17-dione, is only 20% as potent as testosterone when tested with a recombinant yeast bioassay (Lorenzen et al., 2005). Estrone, the only 17ß-estradiol transformation product detected, has approximately half the potency of 17ß-estradiol (Colucci and Topp, 2002). Overall, factors that hasten the transformation of testosterone or 17ß-estradiol such as organic amendment will significantly reduce the androgenic and estrogenic activity, and therefore risk, following application to soil.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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-androstan-3,17-dione, and 1,4-androstadiene-3,17-dione, and 17ß-estradiol to estrone. Biosolids and swine manure slurry inhibited the mineralization of [14C]-testosterone, and stimulated the mineralization of [14C]-17ß-estradiol. Differential sterilization experiments suggested that swine manure slurry carried microorganisms that converted testosterone and estrone to steroidal transformation products, whereas mineralization of [14C]-testosterone required viable soil microorganisms. Similarly, microorganisms carried in swine manure slurry hastened the conversion of 17ß-estradiol to estrone in manured soils, and mineralization of [14C]-17ß-estradiol required soil microorganisms. The observed amendment effects were qualitatively consistent, but quantitatively variable according to soil temperature, soil type, amendment concentration, and particular amendment sample. Under all conditions, hormonal parent compounds and transformation products were completely dissipated within a few days. Overall, swine manure slurry and municipal biosolids, organic matrices that can carry androgenic or estrogenic hormones, hastened the conversion of testosterone and 17ß-estradiol to less hormonally active transient products in agricultural soil. |
ACKNOWLEDGMENTS |
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REFERENCES |
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