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Mineralization of Hormones in Breeder and Broiler Litters at Different Water Potentials and Temperatures

^a Institute of Ecology
^b Department of Crop & Soil Sciences, 3111 Plant Sciences, University of Georgia, Athens, GA 30602-7272

^* Corresponding author (pghartel{at}uga.edu)

Received for publication August 5, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
When poultry litter is landspread, steroidal hormones present in the litter may reach surface waters, where they may have undesirable biological effects. In a laboratory study, we determined the mineralization of [4–¹⁴C]-labeled 17ß-estradiol, estrone, and testosterone in breeder litter at three different water potentials (–56, –24, and –12 MPa) and temperatures (25, 35, and 45°C), and in broiler litter at two different water potentials (–24 and –12 MPa) and temperatures (25 and 35°C). Mineralization was similar in both litters and generally increased with increasing water content and decreasing temperature. After 23 wk at –24 MPa, an average of 27, 11, and <2% of the radiolabeled testosterone applied to breeder litter was mineralized to ¹⁴CO₂ at 25, 35, and 45°C, respectively. In contrast, mineralization of the radiolabeled estradiol and estrone was <2% after 25 wk at all water potentials, except after 17 wk at 25°C and –12 MPa, where up to 5.9% of the estradiol and 7.8% of the estrone was mineralized. The minimal mineralization suggests that the litters may still be potential sources of hormones to surface and subsurface waters.

	INTRODUCTION

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POULTRY LITTERS contain appreciable amounts of the hormones 17ß-estradiol (henceforth, estradiol) and testosterone (Shore et al., 1993; Lorenzen et al., 2004). When the litters are landspread, they can contribute significant amounts of these hormones to surface waters via runoff, depending on application rate and onset of rainfall (Finlay-Moore et al., 2000), amendment with alum (aluminum potassium sulfate; Nichols et al., 1997), and the length of the buffer strip around the amended grassland (Nichols et al., 1998). Although testosterone can move through soil (Casey et al., 2004), estradiol may (e.g., Peterson et al., 2000) or may not (Casey et al., 2003) move in a similar fashion. Study of these hormones is important because they are responsible for the development of secondary sex characteristics in humans and other vertebrates, and they, as well as their metabolites (e.g., estrone), can have undesirable biological effects in surface waters (Lai et al., 2002; Seki et al., 2004). These hormones are also important because of the sheer volume of poultry litter that is applied to land. For example, assuming a litter production rate of 1.46 kg per broiler (Perkins et al., 1964), Georgia's 1.3 billion broilers (Georgia Agricultural Statistics Service, 2005) generated almost 1.9 million Mg of litter in 2004. Most poultry litter is landspread as part of good agronomic practice (Moore et al., 1995).

Concentrations of hormones in poultry litters vary depending on the sex and the type of bird. Broiler litter contains 14 and 65 µg of "estrogen" (likely both estradiol and its main bioactive metabolite, estrone) kg^–1 of litter from male and female broilers, respectively, and 133 µg of testosterone kg^–1 of litter from both sexes (Shore et al., 1993). Breeder litter contains more than eight times more estrogen (533 µg kg^–1 of litter) and almost twice as much testosterone (254 µg kg^–1 of litter) compared to litter from female broilers (Shore et al., 1993). However, lower concentrations of these two hormones have also been observed. Lorenzen et al. (2004) determined that broiler litter contains approximately 55 µg of estradiol and 30 µg of testosterone kg^–1 from both sexes, and that breeder litter contains approximately 70 µg of estradiol and 23 to 30 µg of testosterone kg^–1.

The degradation of estradiol and testosterone varies in different media. Aerobic composting of poultry litter reduces concentrations of estradiol (half-life, 69 d) and testosterone (half-life, 46 d; Hakk et al., 2005), whereas fermenting poultry litter reduces estradiol concentrations but increases testosterone concentrations (Shore et al., 1993). In agricultural soils, estradiol is rapidly converted to estrone (where the hydroxyl at C-17 is oxidized to a carbonyl group), and both are slowly mineralized (12 to 17% is recovered as ¹⁴CO₂) after 3 mo (Colucci et al., 2001). Aside from estrone, other metabolites of estradiol are not commonly observed (Jacobsen et al., 2005). In contrast, half of applied ¹⁴C-testosterone in agricultural soil is mineralized to ¹⁴CO₂ after 120 h, while the remainder is present as three major metabolites: 4-androstene-3,17-dione; 5-androstane-3,17-dione; and 1,4-androstadiene-3,17-dione (Lorenzen et al., 2005). In all cases, mineralization implies cleavage of the steroid backbone with deactivation of the hormone and a concomitant release of CO₂ (Layton et al., 2000).

Although it is standard practice to remove caked poultry litter from the poultry house floor between flocks, the remaining litter can be left in place for 1 to 2 yr before the house is completely cleaned out (Ritz et al., 2005). Mineralization of estradiol, estrone, and testosterone under these conditions has not been identified. Therefore, we conducted a study to determine the mineralization of these three hormones in fresh breeder and broiler litters. Mineralization (as CO₂) was determined with [4–¹⁴C]-labeled hormones. Because estradiol, estrone, and testosterone mineralization is affected by temperature and water potential in soil (Colucci et al., 2001; Lorenzen et al., 2005), our studies were conducted at three different water potentials (–56, –24, and –12 MPa; equivalent to approximately 150, 250, and 350 g of water kg^–1, respectively) and temperatures (25, 35, 45°C) typical of a poultry house floor. A temperature of 45°C was included because the narrow C to N ratio of poultry litters may cause them to heat spontaneously (Hartel et al., 2000). Fresh broiler litter with and without alum was also tested because alum is often added to poultry litter to control the pH (which reduces ammonia volatilization) and litter moisture (Ritz et al., 2005), and alum significantly reduces estradiol in runoff (Nichols et al., 1997). Composting litter reduces, but does not eliminate, estradiol and testosterone (Hakk et al., 2005); therefore, composted litter was also included as a treatment.

	MATERIALS AND METHODS

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Sample Collection and Processing
Four samples of litter were obtained from inside commercial poultry houses located in Gordon and Jackson Counties, Georgia: (i) fresh (<2 wk after bird removal) breeder litter without alum, (ii) fresh broiler litter amended with alum at a commercial rate (20 g of alum kg^–1 litter), (iii) fresh broiler litter without alum, and (iv) composted broiler litter. None of the fresh litters had been stacked; the composted litter had been stacked and composted (temperature > 45°C; C. Ritz, personal communication, 2004). Each sample was homogenized by mixing in a polyethylene bag, and the litter was passed through a 4-mm sieve. The pH and N (total, NH₄⁺–N, and NO₃^––N), P, and K content of the litters were determined as described by Peters (2003) (Table 1). Briefly, the pH was determined in a slurry of litter and distilled water (1:2, v/v). Total N was determined by dry combustion (LECO, St. Joseph, MI), and NH₄⁺–N and NO₃^––N were determined by distillation and titration. Phosphorus and K were determined by digesting the litter in hot HNO₃ and analyzing the digest with inductively coupled plasma spectroscopy. The water potential of each litter was determined at 25°C with a dewpoint potentiameter (Model WP4-T; Decagon, Pullman, WA), which measures both matric and osmotic water potentials. The percentage water content of each litter was determined gravimetrically by drying a portion of the litter at 60°C for 24 h.

Mineralization Studies
Mineralization studies were conducted with [4–¹⁴C]-labeled estradiol, estrone, and testosterone (all approximately 2.00 GBq mmol^–1; purities > 99%; American Radiolabeled Chemicals, St. Louis, MO). For breeder litter, a 200-g portion of each litter was oven-dried at 60°C, and was passed through a 2-mm sieve. The dried litter was divided into ninety 1.0-g samples, each contained in a 50-mL beaker. A 40-µL portion each of radiolabeled estradiol, estrone, and testosterone was diluted separately in 9.0 mL ethanol contained in an ice bath (to minimize ethanol evaporation) such that each stock contained approximately 16.44 kBq mL^–1. A 300-µL aliquot of the ethanolic stock solution was added to each of 30 beakers, and the litter was allowed to air dry at room temperature for 1 h. The radiolabeled litter was mixed thoroughly into 9.0 g of litter (wet weight) with a glass rod, and deionized water was added to bring each sample to 10.0 g at the appropriate water potential. These hormone amendments are similar to those used in other studies (e.g., Jacobsen et al., 2005). Each beaker was placed in a wide-mouth Mason jar with a 20-mL glass scintillation vial containing 2 mL of fresh 1 M KOH (to trap ¹⁴CO₂). Each jar was tightly sealed and placed in an incubator at 25, 35, or 45°C. The error for each incubator was ±2.0°C. In addition, jars containing unlabeled litter for each treatment (negative control) were added to the experiment. Broiler litter was treated in the same way as breeder litter, but the study was conducted only at 25 and 35°C. The broiler litter with and without alum, as well as the composted litter, were incubated only at 25°C.

Except for breeder litter, CO₂ traps were removed for counting and were replaced on Days 2, 4, 8, 15, then approximately weekly thereafter for 13 wk. This length of sampling was selected because it is similar to other hormone mineralization studies (Colucci et al., 2001). In the case of breeder litter, the litter was incubated an additional 10 to 12 wk because the beginning of estradiol and estrone mineralization was observed. Water potential of the litter was maintained by weighing each 50-mL beaker every 2 wk and adding deionized water as needed. At each sampling, 18 mL of ScintVerse cocktail (Fisher Scientific, Hampton, NH) was added to each scintillation vial, and the radiolabel measured in a liquid scintillation counter (Model LS 6500; Beckman Coulter, Fullerton, CA). Quenching was corrected with an external standard.

Litter Oxidation
To confirm the presence of radiolabeled hormone in each beaker, two 0.1-g wet weight portions of litter from each beaker (for a total of six samples per treatment) were combusted in a biological oxidizer (Model OX500; R.J. Harvey Instrument Corp., Hillsdale, NJ). Beakers were removed from the Mason jars at the end of each study, placed in a polyethylene bag, and frozen at –18°C until the analysis. Carbon dioxide was captured in Carbon-14 cocktail (R.J. Harvey), and the radioactivity measured in the liquid scintillation counter. Blank (unlabeled) 0.1-g portions of litter and standards spiked with known amounts of radiolabeled hormone were also oxidized. Finally, one entire 10-g sample of ¹⁴C-hormone-amended broiler litter was divided in 0.1-g portions and was oxidized to determine the distribution of radiolabel in the litter. The inside of the 50-mL beaker was rinsed three times with 2 mL of ethanol, and the rinsate measured in the liquid scintillation counter to recover any radiolabel on the glassware.

Statistical Methods
Treatments were arranged in randomized blocks, and there were three replicates for each treatment. Data for breeder and broiler litter were analyzed with SAS (Release 8.2; SAS Institute, 2001) using a three-way ANOVA at each measurement occasion. Non-constant variance was adjusted using a power of the mean variance function with distinct power parameters fitted for each hormone.

	RESULTS

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Generally, mineralization increased with decreasing temperature and increasing water content. In breeder litter, testosterone was mineralized more than estradiol and estrone regardless of temperature or water potential. An average of <2% testosterone was mineralized at 45°C at both –24 and –12 MPa (Fig. 1B and 1C). At 35°C, testosterone mineralization increased to 10.5% at –24 MPa and 12.6% at –12 MPa (Fig. 1E and 1F). At 25°C, testosterone mineralization was highest, with 27% of the [4–¹⁴C]-testosterone initially applied to the breeder litter converted to ¹⁴CO₂ at –24 MPa, and 21% mineralized at –12 MPa (Fig. 1H and 1I). Results at –56 MPa were not considered after 5 wk because the litter absorbed atmospheric moisture and the treatment was similar to –24 MPa after this time. Unsurprisingly, the –56 MPa results were similar (<2% testosterone mineralized at 45°C, 4% at 35°C, and 31% at 25°C; Fig. 1A, 1D, and 1G) to the –24 MPa results after 5 wk. At 25°C, the lag phase to begin testosterone mineralization decreased with increasing water content (5 wk at –56 MPa, 3 wk at –24 MPa, and <1 wk at –12 MPa).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Mineralization of [4–14C]-labeled estradiol (closed triangles), estrone (open squares), and testosterone (closed circles) in breeder litter at three different temperatures (25, 35, and 45°C) and water potentials (–56, –24, and –12 MPa). Standard error is shown where the error bar is larger than the symbol. Vertical dashed line indicates point at which the –56 MPa treatment was equivalent to the –24 MPa treatment.

In breeder litter, both the type of hormone and the temperature were highly significant variables (p < 0.001) at every measurement occasion, whereas water potential was only highly significant for the first 5 wk and significant (p < 0.05) for the last 3 wk. As expected, significant interactions were observed between hormone and temperature, and, separately, between hormone and water potential at the same measurement occasions. Significant three-way interactions among hormone, temperature, and water potential were also observed for the first 5 wk and the last 8 wk.

In broiler litter, mineralization of estradiol, estrone, and testosterone was similar to breeder litter at 13 wk (figure not shown). At 35°C, 11% of testosterone was mineralized at –24 MPa, and 7.0% of testosterone was mineralized at –12 MPa. Testosterone mineralization was highest at 25°C, with 16% mineralized at –24 MPa, and 15% mineralized at –12 MPa. Compared to breeder litter at 35°C, testosterone mineralization was only 6.0% higher at –24 MPa and 1.0% higher at –12 MPa; similarly, at 25°C, testosterone mineralization was only 7.5% lower at –24 MPa and 3.5% lower at –12 MPa. Less than 1% of estradiol and estrone was mineralized in the four broiler litter treatments after 13 wk regardless of water potential and temperature. Therefore, differences in mineralization between breeder and broiler litter were small. Like breeder litter, the type of hormone was a highly significant variable at every measurement occasion for broiler litter, but the temperature became a significant variable only after 6 wk. Two-way interactions were similar to breeder litter, but three-way interactions were significant only between 5 and 7 wk.

In broiler litter amended with alum or left unamended, the type of litter did not significantly affect the percentage and rate of mineralization of estradiol, estrone, or testosterone after 13 wk (Table 2). No significant differences were observed among the three hormones in composted litter, where <0.1% each of estradiol, estrone, and testosterone were mineralized after 13 wk.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Although decreases in water-soluble estradiol and testosterone have been observed in composted poultry manure (Hakk et al., 2005), our study is the first to describe mineralization of hormones in fresh breeder and broiler litters under different water potentials (–56, –24, and –12 MPa) and temperatures (25, 35, 45°C) that might be expected in a poultry house. In all fresh breeder and broiler litter treatments, mineralization of estradiol, estrone, and testosterone was minimal (<30%), but where it did occur, mineralization generally increased with increasing water content and decreasing temperature.

More [4–¹⁴C]-testosterone was converted to ¹⁴CO₂ than estradiol or estrone. This result is similar to what has been observed previously in agricultural soils (testosterone, Lorenzen et al., 2005; estradiol and estrone, Colucci et al., 2001), and in soils amended with biosolids or swine manure slurry (Jacobsen et al., 2005). Also, the rate of testosterone decline is almost twice as fast as estradiol in composted poultry manure amended with clay (Hakk et al., 2005) and in municipal biosolids (Layton et al., 2000). Finally, estrone and estradiol mineralization was similar in all of the litters tested. This result was also expected because estradiol and estrone are mineralized at similar rates in municipal biosolids (Layton et al., 2000).

The most likely explanation for greater testosterone mineralization compared to estradiol was the physiochemical nature of the hormones. Estradiol (and estrone) contain an aromatic A ring, which is difficult for microorganisms to mineralize (Hakk et al., 2005). Also, estradiol and estrone have higher sorption coefficients (K_d), and therefore sorb more strongly to soil (e.g., Casey et al., 2003, 2004) and may be less bioavailable than testosterone. Finally, some metabolites may be less bioavailable than their parent compounds because of higher K_d values. For example, if testosterone was converted to 1,4-androstadiene-3,17-dione, a compound with a higher K_d than testosterone, then it would be expected to be less bioavailable (Jacobsen et al., 2005). This decrease in bioavailability may explain why testosterone mineralization slowed after several months. It is important to note that only ¹⁴CO₂ and ¹⁴C-labeled, litter-bound residues were measured here and intermediate metabolites of testosterone were not distinguished. In contrast the mineralization of estrone, the major metabolite of estradiol, was distinguished.

Given the high K_d values of the hormones, the compounds should be tightly sorbed to the organic matter in poultry litters. Indeed, other studies show that sorption of estradiol and testosterone increases with substrate organic matter content and surface area (e.g., Lai et al., 2000); only 52% of estradiol and 62% of testosterone spiked into composted poultry litter is extractable with water after 2 h (Hakk et al., 2005). This sorption may also partly explain why the total amount of mineralization observed in the poultry litters in this study was lower than that observed in soil. In agricultural soils, 50% of applied testosterone was mineralized after 120 h (Lorenzen et al., 2005) and 10 to 17% of applied estradiol was mineralized after 3 mo (Colucci et al., 2001). Here, even under optimal conditions, the total amount of testosterone mineralized was <30% after 23 wk, and the total amounts of estradiol and estrone mineralizedwere <10% after almost double the time of the soil study.

Water content and temperature may also explain why the total amount of mineralization observed in the poultry litters was minimal in this study. In terms of water potential, estradiol and testosterone dissipation decreases in soils with decreasing water content (Colucci et al., 2001; Lorenzen et al., 2005), and a water potential of –12 MPa is sufficient to limit much microbial activity (Harris, 1981). Indeed, litter should be kept at a water potential less than –12 MPa to limit the proliferation of pathogens (Ritz et al., 2005). In this study, the increasing lag phase to mineralize testosterone in the breeder litter with decreasing water content may also reflect the effect of water potential on microbial activity.

In terms of temperature, estradiol dissipates faster in soils at temperatures between 30 and 37°C than at 4 and 10°C (Colucci et al., 2001); similarly, testosterone dissipates faster in soils at 23 and 30°C than at 4 and 12°C (Lorenzen et al., 2005). Although our study did not measure mineralization at such low temperatures, on a statistical basis, temperature was the most significant variable affecting hormone mineralization in our study, and mineralization generally decreased as temperature increased. The differences in mineralization with temperature observed in our study compared with the results in other studies may reflect differences between litter and soil. Given that a low water potential (less than –10 MPa; common in poultry litter and uncommon in soils) favors fungi (Harris, 1981), it may be that the fungi that biodegrade hormones in litter are mostly mesophiles, not thermophiles. Therefore, even though stacking litter, a passive form of composting where temperatures are in the range tested here (45°C), may kill microbial pathogens (Hartel et al., 2000), it may have little or no effect on hormone content.

Mineralization in broiler litter was similar to breeder litter, with the highest mineralization rates for testosterone at 25°C, and <1% of the estrogens mineralized in 13 wk. Because breeder litter contains higher estrogen and androgen concentrations compared to broiler litter (e.g., Shore et al., l993), and microbes may be adapted to these higher concentrations, greater mineralization of the hormones was expected in breeder litter. For reasons that are unclear, this greater mineralization did not occur. Even so, it is unlikely that hormone mineralization will be the same in all types of poultry litter, because the litter may be stacked (Hakk et al., 2005) or the feces in some litters may contain antibiotics. For example, when antibiotics were added to broiler litter and the litter fermented, estrogen degradation was inhibited, whereas testosterone concentrations increased (Shore et al., 1993). Amending broiler litter with alum had no significant effect on the mineralization of estradiol, estrone, or testosterone. Therefore, adding alum to reduce estradiol in runoff (Nichols et al., 1997) is unlikely to affect hormone mineralization. In composted litter, no significant amounts of estradiol, estrone, and testosterone were mineralized, even though composting reduces water-soluble concentrations of estradiol by 84% and testosterone by 90% (Hakk et al., 2005). The most likely reason was the low pH of the composted litter (pH 5.2), which would have reduced microbial activity (Hartel et al., 2000). Jacobsen et al. (2005) show that mineralization of testosterone and estradiol requires viable microorganisms to proceed.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
This study is the first to describe mineralization of estradiol, estrone, and testosterone in breeder and broiler litters under conditions likely to be encountered in a poultry house. Depending on the temperature and water potential, the compounds were either not mineralized or mineralized only to a limited extent. Under optimal conditions, 5.9% of the estradiol and 7.8% of the estrone were mineralized after 25 wk, and 27% of the testosterone was mineralized after 23 wk. Adding alum to the litter did not affect estradiol, estrone, and testosterone mineralization significantly. The limited mineralization suggests that the litters may still be potential sources of hormones to surface and subsurface waters, where they may have undesirable biological effects. Future studies are needed to evaluate the mineralization of these hormones once poultry litter is applied to soils.

	ACKNOWLEDGMENTS

 
We thank Jared Fisher, Wendy Giminski, Travis Hanselman, Ray Hemmings, Casey Ritz, Karen Rodgers, Hathai Sangsupan, William Vencill, Paul Vendrell, and Ann Wilkie for their technical assistance, and Miguel Cabrera, Ron Carroll, and Kang Xia for their comments on the manuscript. Also, we thank Daniel Hall and Jing Xu of the University of Georgia Statistical Consulting Service for their statistical analysis. This research was partially supported by grants from the University of Georgia Foundation Fellowship and the University of Georgia Graduate School.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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