Testosterone

Biodegradation in soil

Administrative data

Endpoint:

biodegradation in soil

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Materials and methods

Principles of method if other than guideline:

The goal of this
research was to investigate the fate and occurrence of
17b-estradiol and testosterone in agricultural soils under
aerobic and anaerobic conditions using incubation experiments.

GLP compliance:

not specified

Test type:

laboratory

Test material

Test material information

Constituent 1

Reference substance name:

Testosterone

EC Number:

200-370-5

EC Name:

Testosterone

Cas Number:

58-22-0

Molecular formula:

C19H28O2

IUPAC Name:

17-hydroxyandrost-4-en-3-one

Details on test material:

[4-14C]-radiolabeled 17b-estradiol and testosterone (>99% by TLC) were purchased from American Radiolabeled Chemicals, St. Louis, MO, and stored at 4 C before use.

Radiolabelling:

yes

Study design

Details on experimental conditions:

The persistence and fate of 17b-estradiol and testosterone were each investigated under the following four soil microcosms: native soil under aerobic conditions, native soil under anaerobic conditions, autoclaved soil under aerobic conditions, and autoclaved soil under anaerobic conditions.
Autoclaved soils were prepared by autoclaving the soils for 40 min at 122 C.

To create a solution concentration of 0.052 mg /l for testosterone and 1.5 mg /l for 17b-estradiol, 75μg of [4-14C]-radiolabeled 17b-estradiol (approximately 1000 000 dpm) or 2.62 μg of [4-14C]-radiolabeled testosterone (approximately 1000000 dpm) per 200 g of soil in approximately 50 ml of 0.01 M CaCl2 (Sigma-Aldrich, St. Louis, MO) was gradually added to the native and autoclaved soils, which adjusted the soil moisture content to 100% waterholding capacity (saturated soil but not slurries) at the beginning of the incubation. These concentrations were chosen because they have been found in animal manures applied to agricultural fields (Shore and Shemesh, 2003; Lorenzen et al., 2004). The weak salt solution (i.e., 0.01 M CaCl2) was used so soil aggregates would not be dispersed. The volumetric fraction of methanol in each initial stock solution was less than 1%, which has been previously shown to not significantly affect the sorption of organic contaminants to soil (Wauchope and Koskinen, 1983). In addition, 500 mg HgCl2 (Fisher Chemical, Fairlawn, NJ) per kg soil was added to the autoclaved soils to inhibit any potential airborne bacterial activities (Trevors, 1996).

About 210 g of soil, not sieved or air-dried, was added to a 250 ml glass flask (Fig. 2). The flask was then covered with aluminum and sealed with a rubber stopper to eliminate the possibility of photodegradation. Tygon tubing (6 mm inner diameter) was used for all connections. Moist air was used as the carrier gas under aerobic conditions, and humidified helium gas was used as the carrier gas under anaerobic conditions. The carrier gas was humidified to maintain a constant soil moisture in either aerobic or anaerobic conditions. In the aerobic systems (Fig. 2), the outlet gas from the soil-filled flask was passed through a tube that was connected to a 200 ml glass flask containing 120 ml of 3 M NaOH, which trapped 14CO2. In the anaerobic systems (Fig. 2), the outlet gas from the soil-filled flask was first passed through a hand-packed Porapak column (i.d. 0.5 cm, length 6 cm), which trapped the 14C-labeled volatile organic compounds, except 14CH4. After passing through the Porapak column, the outlet gas continued through two traps. The first trap contained 120 ml of Bray’s solution, which trapped 14CH4, and the second trap contained 120 ml of 3 M NaOH, which trapped 14CO2. On hours 1, 2, 3, 4, 5, 30, 54, and 132, two 500 ll aliquots were removed from each of the NaOH and Bray’s solutions and were dissolved in 16 ml of scintillation cocktail, allowed to settle for at least 48 h at room temperature, and assayed for radioactivity by liquid scintillation counting (LSC; 1900 CA scintillation counter, Packard, Downers Grove, IL). At 132 h, the experiment was stopped and the Porapak MeOH, and 3 ml of acetone. Aliquots of 100 ll were removed from each of these sequential washings and assayed for radioactivity by LSC.

Additionally, extractability of resident 14C in the soil was determined. This was accomplished by extracting the soil first with water (three times the soil moisture content), and then with acetone (three times the soil moisture content). Aliquots of 500 ll were removed from the two extracts and assayed for radioactivity by liquid scintillation counting (LSC). Analysis for metabolites was also done on the two extracts using thin-layer chromatography (TLC) (System 2000 Imaging Scanner (Bioscan, Inc., Washington DC)). The TLC was done using silica gel plates (250 lm; Whatman Lab. Div., Clinton, NJ) developed with methylene chloride:ether: hexane (2:3:4).

Following the solvent extraction, the remaining nonextractable 14C was fractionated as described by Kaplan and Kaplan (1982) to determine the distribution of nonextractable 14C among the various organic matter fractions (e.g., humic acid, fulvic acid, and humin). This was done in the following order: (1) air-drying and grinding the soil, (2) placing 50 g of dry soil into a 500 ml cylinder, (3) washing the soil with 200 ml of 0.1 N HCl, (4) extracting the soil with 200 ml of 0.5 N NaOH and 0.1 N Na4P2O7 Æ10H2O, respectively, (5) combining the two fractions of NaOH and Na4P2O7 Æ10H2O, and (6) adjusting the combined solution to pH 3.0. The insoluble materials that remained after steps (4) and (6) were considered to be associated with humin and humic acid, respectively. The radioactivity associated with these two organic matter fractions (i.e., humin and humic acid) were assayed by combustion analysis on a Packard Model 307 Oxidizer (Packard Chemicals, Meridan, CT). The soluble material after step (6) was considered to be associated with fulvic acid, and was assayed for radioactivity by LSC.

Applicant's summary and conclusion

Conclusions:

The TLC results also indicated that testosterone was degraded in soils, not by physical– chemical processes but by biological processes.

Executive summary:

Steroidal hormones are constantly released into the environment by man-made and natural sources. The goal of this study was to examine the persistence and fate of testosterone in soils. Incubation experiments were conducted under aerobic and anaerobic conditions using [4-14C]-radiolabeled testosterone. The results indicated that 63% of testosterone could be mineralized to 14CO2 in native soils under aerobic conditions. In native soils under anaerobic conditions, 2% of testosterone was methanogenized to 14CH4. Essentially, no mineralization of testosterone to 14CO2 occurred in autoclaved soils under aerobic or anaerobic condition.