Environ. Sci. Technol. 2004, 38, 790-798

Fate and Transport of Testosteronein Agricultural Soils
F R A N C I S X . M . C A S E Y , * ,†H E L D U R H A K K , ‡ J I Ř IÄ Š I M U° NEK,§ AND
GERALD L. LARSEN‡
Department of Soil Science, North Dakota State University,
Fargo, North Dakota 58105;Animal Metabolism
Agrochemicals Research Unit, Biosciences ResearchLaboratory, USDA-ARS, Fargo, North Dakota 58105; andDepartment of Environmental Sciences, University ofCalifornia, Riverside, California 92521

Hormones excreted in animal waste have been measured
in surface and groundwater associated with manurethat is applied to the land surface. Limited studies havebeen done on the fate and transport of androgenic hormonesin soils. In this study, batch and column experiments
were used to identify the fate and transport of radiolabeled[14C] testosterone in agricultural soils. The batch results
indicated that aqueous-phase concentrations decreased forthe first 5 h and then appeared to increase throughtime. The first-order sorption kinetics ranged from 0.08 to
0.640 h-1 for the first 5 h. Beyond 5 h the increase inaqueous 14C could have been caused by desorption of
testosterone back into the aqueous phase. However,
metabolites were also produced beyond 5 h and would
have likely resulted in the increase in aqueous 14C by sorption
site competition and/or by lower sorption affinity. There
were weak correlations of sorption with soil particle size,organic matter, and specific surface area. Testosterone
was the dominant compound present in the soil column
effluents, and a fully kinetic-sorption, chemical nonequilibrium
model was used to describe the data. Column experiment
sorption estimates were lower than the batch, which
resulted from rate-limiting sorption due to the advective
transport. The column degradation coefficients (0.404-0.600h-1) were generally higher than values reported in theliterature for 17β-estradiol. Although it was found that
testosterone degraded more readily than 17β-estradiol, it
appeared to have a greater potential to migrate in the soil
because it was not as strongly sorbed. This study
underlined the importance of the simultaneous transformationand sorption processes in the transport of hormones
through soils.

Introduction
Documented cases of reproductive and developmental
abnormalities in numerous vertebrate and invertebrate
animal species have been identified over the last few decades(1). It has been implied that endocrine-disrupting chemicals(EDCs), such as reproductive hormones (i.e., testosterone
and 17β-estradiol), are the cause of these abnormalities. A* Corresponding author phone: (701) 231-8577; fax: (701) 2317861; e-mail: francis.casey@ndsu.nodak.edu.
† North Dakota State University.
‡ USDA-ARS.
§ University of California, Riverside.
790 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 3, 2004

recent survey of 139 streams across 30 states in the UnitedStates has detected reproductive hormones in approximately
40% of the streams sampled (2). The sources of thesechemicals are industrial, agricultural, and residential andcan cause reproductive abnormalities in aquatic species at
extremely low levels (e.g., <1 ng L-1) (3). Some suggest thata threshold of exposure does not exist for EDCs because they
mimic or antagonize the actions of an endogenous molecule(4). Adverse responses to EDCs in the environment are
escalated if the EDC is the actual endogenous molecule, suchas a hormone or synthetic hormone.
Hormonal EDCs are found and released in the environment and can result in adverse health impacts to aquaticorganisms. Renner (5) recently reported that hormoneadulterated runoff from cattle (Bos Taurus) feedlots could
have androgenic effects on local female fish, which havemanifested male characteristics. Natural and synthetic
hormones are given to cattle to enhance growth by increasing
the efficiency of converting feed to meat. Manure that containthese synthetic growth hormones (concentration ranging
from 6 to 10 µg kg-1) are applied to soils as fertilizer and aretraceable in the soil up to 8 weeks after fertilization (6).
Testosterone is also produced naturally by poultry (Gallus
gallus domesticu) and has been measured in their manure
at average concentrations of 133 µg kg-1 for female broilers
and 670 µg kg-1 for male roosters (7). Almost 12 million Mgof broiler litter is produced in the United States each year,
which contains natural levels of testosterone, and almost all
the litter is applied to soil as fertilizer. Hormonal treatments
are also used in aquaculture operations to manipulate fish
phenotypic sex to produce a monosex population (8).
Synthetic androgenic hormones are provided in fish feed indoses between 0.5 and 5 mg kg-1 of feed. If EDCs are, assome suggest, the cause of widespread adverse healthimpacts, then what are the possible exposure routes to thesechemicals?
Limited studies have reported on the fate and transportof androgenic hormones in the environment. Shore et al. (9)measured testosterone concentrations in an irrigation pond
and a local stream near a field that received poultry litter(containing 34 µg kg-1 testosterone). The concentration of
testosterone in the irrigation pond ranged from 0.5 to 5 ng
L-1, and the concentrations from the stream ranged from 1
to 28 ng L-1. Another study by Finlay-Moore et al. (10)measured testosterone in soil and runoff from grasslands
amended with broiler litter. Runoff concentrations of testosterone ranged between 10 and 1830 ng L-1 and depended
on manure application rate and frequency. Following manure
application, the soil testosterone concentrations increased
to approximately 150-790 ng kg-1 from background concentrations (15-55 ng kg-1). In some cases, the soil testosterone concentrations persisted or increased within (andperhaps beyond) a 14 day period after manure application.
Finlay-Moore et al. (10) suspected possible movement of
testosterone below their soil sampling depths of 0-2.4 cm
and made conclusions similar to Shore et al. (9), whosuggested that testosterone leached more readily than theprinciple estrogenic hormone, 17β-estradiol. 17β-Estradiol
has been found at significant concentrations (ranging from
6.0 to 66.9 ng L-1) in springs that are fed by aquifers underlying
areas associated with poultry manure application (10, 11).
The concentrations of the hormone in the springs closely
paralleled the levels of fecal coliform associated with animal
manures. In areas associated with manure application,
testosterone may be of equal or greater risk to subsurface
water quality compared to 17β-estradiol.
10.1021/es034822i CCC: $27.50

 2004 American Chemical Society
Published on Web 12/16/2003

TABLE 1. Soil Fractions Used for Experiments, Their Official Soil Series Description, Organic Matter Content, and Specific Surface
Measurement
soil series

taxonomic description

Bearden-silty clay loam
Gardena-clay loam
Glyndon-sandy clay loam
LaDelle-silt loam
Sioux-loamsandfine-silty, mixed, superactive, frigid Aeric Calciaquolls
coarse-silty, mixed, superactive, frigid Pachic Hapludollscoarse-silty, mixed, superactive, frigid Aeric Calciaquolls
fine-silty, mixed, superactive, frigid Cumulic Hapludolls
sandy-skeletal, mixed, frigid Entic Hapludolls

Limited studies have reported on the fate and transportof androgenic hormones in soils. Layton et al. (12) measured
mineralization of testosterone by biosolids in a wastewater
treatment system, yet little is known about degradation/transformation and mobility in soils. The objectives of thisstudy were to investigate the sorption, transformation, andmobility of testosterone in agricultural soils. Testosterone
was chosen because it is produced by all studied animal
species and thus has the potential of being widespread; it ishighly potent and can metabolize to produce other potent
metabolites; it is a prototype for other synthetic androgenic
hormones; and little is known about the fate and transportof androgenic hormones. Miscible-displacement and batch
sorption experiments were used to identify the fate andtransport mechanisms in soils ranging in particle size
distributions and organic matter content. Inverse modelingtechniques were used with the column studies to help identify
transport processes. The experiments were designed usingsoils and application rates associated with agricultural
practices that include manure fertilization.

Materials and Methods
The materials and methods that were used for the batch andcolumn studies were nearly identical to an earlier study that
identified the fate and transport of 17β-estradiol (13). The
upper soil horizon, an A-horizon, of a Bearden-silty clay loam,
Gardena-clay loam, Glyndon-sandy clay loam, LaDelle-silt
loam, and Sioux-loam (Table 1) were used for the batch andmiscible-displacement experiments. These soils were obtained from Ag-Vise Company (Northwood, ND) except the
Glyndon. These soils are formed under prairie, predominantly
found in North Central United States, and are typically highin organic matter. They also represent a broad range of soil
textures. Additionally, a quartz sand (250-500 µm) was used
for the batch and column experiments. All the soils were
initially dried at 85 °C for 24 h. Major physical and chemical
properties of each soil type were measured at the Soil andWater Environmental Laboratory at North Dakota State
University. These measurements included particle size
distribution (14) and organic matter content (15) (Table 1).
Specific surface area was also measured using the ethylene
glycol monoethylene ether method (16).
Equilibrium Batch Sorption Experiments. Soil and water(0.0l M CaCl2) were added to 10 mL vials in a ratio of 1.6 g
to 8 mL, respectively. The batch equilibrium experiments
were done using concentrations of 14C radiolabeled testosterone (American Radiolabeled Chemicals, St. Louis, MO),
which was added to triplicate vials to create solution
concentrations of 0.725, 0.399, 0.0725, and 0.00725 µg mL-1.
These concentrations were chosen because they span therange of concentrations reported for manures that are applied
to fields (7, 9, 10). The soil-water slurries were agitated byrotation of the vials top to bottom (360°/5 s).
After 0.5, 1, 5, 24, 48, 96, and 168 h, the bottles were
centrifuged at 1700 rpm (1000g), triplicate 100 µL aliquots
were removed and assayed for radioactivity by liquid
scintillation counting using a 1900 CA Scintillation Counterorganic matter
content (%)

specificsurface (m2 g-1)

7.5
5.3
3.3
9.2
7.5
0.0

175
154
123
151
106

(Packard, Downers Grove, IL). Also, thin-layer chromatography (TLC) [System 2000 Imaging Scanner (Bioscan, Inc.,
Washington, DC)] was used to determine if transformation
occurred by identifying the presence of metabolites. Thinlayer chromatography (TLC) analysis on these fractions was
conducted using silica gel plates (250µm; Whatman Lab. Div.,
Clinton, NJ) with the following solvent system: tetrahydrofuran:ethylacetate:hexane (12.5:12.5:25). A combustion analysis assay [Packard Model 307 Oxidizer (Downers Grove, IL)]
was also performed for total 14C sorbed to the soil. Using
these methods, the lowest calculated detection limit for
testosterone was 7.3 ng L-1 in terms of water (8 mL) and 36
ng g-1 in terms of soil (1.6 g).
Freundlich isotherms were used to describe the batch
experiments where the concentration of solute adsorbed on
the soil, S (mg g-1), is related nonlinearly to the aqueousconcentration in the soil solution, C (mg L-1)

S ) KfCn(1)

where Kf (L g-1) is the Freundlich distribution coefficientand n is an empirical constant that controls the deviationfrom linearity (n ) 1 is linear). A nonlinear, least-squares
approximation method (17) was used to obtain the best-fit
of eq 1 to the observed data by optimizing the unknown
parameters, Kf, and n.
Additionally first-order kinetic sorption constants, ω (h-1),
were estimated using the following expression

C(t) ) C0e-ω(t-t0)(2)where t is time and the subscript 0 indicates initial values.
The ω values were estimated by fitting eq 2 to measured dataalso using a least-squares, nonlinear curve-fitting routine(17). The optimized ω value was obtained by changing ititeratively until a best fit of eq 2 to the data was achieved.
Miscible-Displacement Experiments. The sand and eachsoil series were packed into individual columns, and bothchloride anion and testosterone were passed through eachcolumn. Chloride was used as a nonsorbing tracer to identify
the physical transport characteristics of the soil columns.
Table 2 provides the major physical properties of eachcolumn. The soils were evenly packed in glass columns(diameter ) 8.4 cm, length ) 15.0 cm) with stainless steel
end caps. Sandwiched between the soil and the end caps
were several layers of cheesecloth and a 40-mesh stainlesssteel screen, which retained the soil in the column. Glass,
Teflon, and stainless steel were used in the construction tominimize adsorption to the experimental apparatus.
Each column was slowly wetted from the bottom over a
24 h period using a weak salt solution (0.01 M CaCl2). This
reduces the amount of entrapped air and maintains soil
structure. After the column was wetted, flow was establishedfrom the top down using the same 0.01 M CaCl2 solution.
Once steady-state pore water velocity (v (cm min-1)) was
achieved, a pulse of chloride ion tracer (0.05 M CaCl2) wasapplied and eluted with the 0.01 M CaCl2 solution. The
VOL. 38, NO. 3, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 791

TABLE 2. Soil Column Physical Properties
soil series

mass ofdry soil (g)volumetric watercontent (cm3 cm-3)

pore watervelocity (cm min-1)

pore
volume (mL)pulse input
relative PV

Bearden-silty clay loam
Gardena-clay loam
Glyndon-sandy clay loam
LaDelle-silt loam
Sioux-loam
sand

784
695
1126
760
825
1391

0.68
0.64
0.52
0.60
0.60
0.34

0.29
0.35
0.36
0.35
0.36
0.54

569
528
435
500
495
286

0.07
0.08
0.09
0.08
0.08
0.05

TABLE 3. First-Order Kinetic Sorption Coefficients (ω) Determined for Each Initial Aqueous Concentration (C0)asoil typeC0 ) 0.725 mg L-1
ω 0-5 h (h-1)

C0 ) 0.399 mg L-1
ω 0-5 h (h-1)

C0 ) 0.0725 mg L-1
ω 0-5 h (h-1)

C0 ) 0.00725 mg L-1
ω 0-5 h (h-1)

Bearden-silty clay loam
Gardena-clay loam
Glyndon-sandy clay loam
LaDelle-silt loam
Sioux-loam

0.377
0.335
0.640
0.375
0.343

0.388
0.325
0.596
0.375
0.320

0.416
0.318
0.547
0.350
0.226

0.461
0.268
n.d.
0.391
0.077

a Sorption coefficients were estimated for time interval 0-5 h.

effluent was fraction collected every 2 min, and the conductivity of each fraction was measured using a conductivity
meter (Oakton PC 300,Vernon Hills, IL). Table 2 provides thevolumetric water content (θ (cm3 cm-3)), pore volume (PV),and v of each column experiment. The residence times in
the columns ranged between 42 and 52 min. Although thevelocities fell in the range of observed soil water flow, they
were somewhat high. These types of velocities might beobserved after a large rainfall or irrigation event.
Following the chloride ion breakthrough curve experiments, several PVs of the 0.01 M CaCl2 solution were flushed
through the soil column. A 40 mL pulse of 14C testosterone(0.91 µCi; 0.131 µg mL-1) was then applied to the surface ofthe soil column and eluted with the 0.01 M CaCl2 solution
for 7-12 PVs. This input testosterone concentration was
relatively high but realistic. Finlay-Moore et al. (10) uses a
broiler litter application rate of 7.05 Mg ha-1, and Shore etal. (7) reports testosterone levels reaching 1 µg g-1 in broiler
litter. This application rate and testosterone concentration
would have resulted in approximately 2.5 µg of testosterone
applied compared to 5.2 µg used in this study. The column
effluent was fraction collected every 2 min, and each fraction
was analyzed for 14C and metabolites using the liquid
scintillation and TLC methods described earlier.
Additionally, the distribution of resident 14C in the column
was determined. The soil was extruded from each column
in 1-cm increments, then dried and assayed for 14C by
combustion analysis as described earlier. Solution extracts
were then obtained from each 1-cm increment by sequential
elution with toluene, ethyl acetate, and methanol in the cellof an Accelerated Solvent Extractor (model 200; Dionex,
Sunnyvale, CA). Analysis for metabolites was also done onthese extracts using TLC analysis described for the batch
experiments.
Miscible-Displacement Model. The miscible-displacement experiments were modeled with the computer program
HYDRUS-1D, version 2.0 (18). This program uses an inverse
modeling technique to fit the model solution to the observed
data in order to estimate reaction and transport parameters.
The inverse modeling approach uses a least-squares optimization routine to obtain the best-fit model solution anddoes this by iteratively changing model parameters until abest fit is achieved.
A fully kinetic, one-site (19, 20), convective-dispersivemodel with degradation and Freundlich kinetic sorption wasconsidered in describing the miscible-displacement experi792 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 3, 2004

ments. This model assumes that solutes exist in either thesorbed or aqueous phases and that degredation occurs inthe sorbed phase. The following is the partial differential
equation that governs the nonequilibrium chemical transport
for a homogeneous system during one-dimensional, steadystate water flow

∂ 2C
∂S
∂C
∂C
+ Fb ) θvλ 2 - θv - µFbS
∂t
∂t
∂x
∂x

(3)

∂S
) ω[KfCn - S] - µS
∂t

(4)

θ

where C is the aqueous concentration (mg L-1), t is time (h),Fb is soil bulk density (g L-1), S is sorbed phase concentration(mg g-1), λ(cm) is the dispersivity, v is steady-state velocity(cm h-1), x is depth (cm), ω is a sorption rate coefficient(h-1), µ is a first-order degradation/transformation rate
constant (h-1), and Kf (L g-1) and n (-) are Freundlich sorption
coefficients (defined in eq 1). For a stable nonsorbing solute,
such as the chloride anion tracer, it is assumed that S ) 0
and µ ) 0.

Results and Discussion
Batch Experiments. Sorption to the solid phase generally
increased from 0 to 5 h with first-order sorption kinetic
coefficients that ranged from 0.08 to 0.640 h-1 (Table 3). After
5 h the sorbed phase concentrations appeared to steadilydecrease for each soil and initial concentration (Figure 1).
Note that each graph in Figure 1 uses a different verticalscale and that this scale does not start at zero. Thus, observed
desorption was significantly smaller than it may seem after
visual inspection of Figure 1. Lai et al. (21) showed similar
1-5 h kinetic sorption values (0.07-0.37 h-1) for similarsteroidal estrogen hormones in the presence of river andestuary sediments. Lai et al. (21) also found similar sorption
behavior through time, where the various estrogens werefirst sorbed rapidly then sorption decreased. They suggest
the decrease in sorption was caused by estrogen desorption
back into the aqueous phase. The observed decrease in sorbed
concentrations at times >5 h (Figure 1) may have resulted
from the desorption of testosterone back into the aqueous
phase, which was similar to estrogens (21, 22) and otherhydrophobic organics. Lai et al. (21) explains the cause of
estrogen desorption back into solution as a result of organic
matter in the water phase (23, 24). In this study, the increaseFIGURE 1. Time series plots of sorbed testosterone for each initial concentration (C0) of 0.73, 0.40, 0.073, and 0.0073 mg L-1.
of 14C in the aqueous phase after 5 h also corresponded tothe presence of testosterone metabolites, which were indicated by TLC analysis. Competition for sorption sites will
occur if the metabolites have a greater sorption affinity thanthe parent molecule, which could explain the slight decreaseof sorbed concentrations. Also, metabolites that have lower
sorption affinities than the parent testosterone could also
explain this decrease.
Solid-solution distribution ratios (Kd ) S/C, L g-1) were
determined for total 14C concentration at each measurement
time (Figure 2). For each soil, the Kd values increased thentended to decrease through time (Figure 2), where maximum
values occurred between 1 and 5 h. The decrease in Kd after
5 h was likely caused by the production of metabolite
compounds. Xing and Pignatello (25) showed similar decreases in Kd values for competitive sorption between
chlorinated benzenes. At t > 5 h, testosterone metabolites
would compete for sorption sites and/or could have lower
sorption affinity, thus decreasing the Kd through time.
The Kd values were also used to observe any correlations
between sorption and various soil properties (Figure 3). In
order of strength, the correlations that were observed were
between Kd and sand (r2 ) 0.34), clay (r2 ) 0.26), specificsurface (r2 ) 0.22), and organic matter content (r2 ) 0.21).
Similar correlations, albeit stronger, are also found between
various estrogens and organic and mineral fractions of soil(13) and river and estuary sediments (21). These correlations
generally indicate a relation between sorption and surface

FIGURE 2. Time series plot of the solid-solution distribution ratios(Kd) with 95% confidence intervals.
area and/or organic matter content, which reflect thehydrophobic nature of the sorbate (testosterone).
The Freundlich isotherm parameters (Table 4) were
determined using the 5 h data because sorption appeared toreach its maximum at this time. The Freundlich n values
were essentially all equal to 1. Values of n ≈ 1 suggest
interaction between a generally hydrophobic sorbate with ahydrophobic sorbent (e.g., pesticide-organic matter interactions) (26). Additionally, many of the n values were greater
VOL. 38, NO. 3, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 793

FIGURE 3. Relationships between various soil components and the solid-solution distribution ratio (Kd) calculated at the indicatedexperimental times. Also included are the linear regressions and 95% confidence intervals.

TABLE 4. Freundlich Coefficients (Kf, n) Determined at 5 hTime for Each Soil
soil type

Kf (L g-1)nBearden-silty clay loam
Gardena-clay loam
Glyndon-sandy clay loam
LaDelle-silt loam
Sioux-loam
sand

0.15
0.05
1.20
0.10
0.09
0.0005

0.98
1.00
1.00
0.98
1.00
0.94

than 1 for isotherms determined at times beyond 24 h (datanot presented). These results indicated that there wereavailable sorption sites and that the decrease in the amountof sorbed phase concentrations observed in Figure 1 (t > 5h) and the decrease in Kd values in Figure 2 (t > 5 h) were
not caused by decreases in sorption site availability. Rather
the decrease was likely caused by metabolite production or
possibly testosterone desorption as previously discussed.
Miscible-Displacement Experiments. There was 14C
present in all the column effluent for each soil except the
Glyndon-sandy clay loam. The total mass of 14C recoveredfrom within the column and the effluent is presented in Table
5. The mass recovery of 14C ranged between 76% and 85%.
Incomplete recoveries may have been the result of determining sorbed phase concentrations using solvent extractionsof the soil, where complete removal of the sorbed 14C may
not have been achieved. Almost all 14C, about 93%, wasrecovered for the sand column effluent, which meant little
or no retention by the sand. This result indicated that
testosterone sorption was likely caused by interactions with
794 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 3, 2004

organic matter and/or particle size or clay content/type. The
majority of the 14C recovered from the extractions from eachsoil column was found in the upper 1-3 cm of the column
and ranged from 14% to 69% of the total 14C mass applied.
Between 5% and 13% of the 14C was still found in the lower
3 cm of the column, which indicated significant redistribution
with depth.
Low concentrations of 14C were present in the column
effluent fractions. The total radioactivity present in thecolumn effluent could be determined with confidence;
however, reliable determination of the concentrations ofindividual metabolites was not possible. To increase thesensitivity for the TLC analysis, it was necessary to combine
several effluent fractions into a single volume. This made it
possible to visualize the presence of metabolites, but theirquantification was difficult. Certain general conclusions could
nonetheless be made from this information. Furthermore,other studies have identified possible testosterone metabolic
pathways by a crustacean (Neomysis integer (27)) and abacteria (Comamonas testosteroni (28)) and helped to
speculate the identity of the metabolites. The crustacean
study identifies possible testosterone metabolites using TLC,
which was compared to TLC results from this study. The 14C
present in the effluent of the Gardena-clay loam and thesand columns was parent testosterone with no metabolite
present. Very small amounts of metabolites were present in
the effluent of the LaDelle-silt loam and Sioux-loam. One
metabolite of greater polarity than testosterone was foundin the effluent of the LaDelle-silt loam column. The polarityof this metabolite was most similar to androstenedione, but
it could also be dihydrotestosterone (27). The Sioux-loam

TABLE 5. Column Model (eqs 3 and 4) Transport Parametera Estimatessoil typeλ (cm)Kf (L g-1)n

ω (h-1)µ (h-1)% 14C recovery

r2

Bearden-silty clay loam
Gardena-clay loam
Glyndon-sandy clay loam
LaDelle-silt loam
Sioux-loam

6.52 ((0.58)
8.10
1.68
3.55
6.71 ((0.42)

0.050 ((0.053)
0.012 ((0.005)
0.054
0.022 ((0.021)
0.006 ((0.010)

1.28 ((0.16)
1.13 ((0.08)
1.30
1.27 ((0.18)
1.16 ((0.35)

0.613 ((0.205)
0.422 ((0.102)
0.057
0.484 ((0.190)
0.877 ((0.522)

0.404 ((0.029)
0.496 ((0.047)
0.600
0.493 ((0.190)
0.493 ((0.522)

76 [11]b82 [32]85 [0]
82 [24]77 [40]

0.86
1.00
0.98
0.95
0.94

a λ ) dispersivity; K and n are Freundlich sorption coefficients; ω ) first-order sorption rate coefficient, µ ) first-order degradation/transformationfcoefficient. Values inside parentheses represent the (95% confidence interval of the estimated parameter. b Values outside brackets represent
total mass recovery; values within brackets represent mass recovered in the effluent.

FIGURE 4. Soil column breakthrough curves of 14C that show the relative effluent concentrations (C/C0 ) measure concentrated/applied
concentrated) versus time.
had trace amounts of two metabolites; one metabolite washigher in polarity than testosterone, and the other was slightly
lower in polarity. The metabolite of higher polarity was mostsimilar to androstenedione (possibly dihydrotestosterone).
The lower polarity metabolite was most similar to 2Rhydroxytestosterone (possibly β-boldenone or 16β-hydroxytesoterone) (27). All of the 14C present in effluent of theBearden-silty clay loam column was found to be a single
metabolite of greater polarity than testosterone, most similar
to androstenedione (possibly dihydrotestosterone) (27). The
TLC analysis of the soil extraction from within each soilcolumn indicated that testosterone and two other metabolites
were present. One metabolite had a higher polarity thantestosterone (either androstenedione or dihydrotestosterone),and the other had a lower polarity (either hydroxytestosterone, 16β-hydroxytesoterone, or β-boldenone) (27). In our
speculation of the testosterone metabolites, it appeared that
all the metabolites that were found in the effluent and within
the column retained their steroidal structure. It is also possiblethat the metabolites could act as endocrine disruptors but

at a reduced potency (27). Unlike 17β-estradiol (13), testosterone was able to elute from the soil columns intact andin some cases was the only compound present in the effluent.
Miscible-Displacement Model Analysis. The asymmetricsolute breakthrough curves (Figure 4), caused by solute tailingor late arrival of solute, are characteristic of nonequilibriumtransport. Physical and/or chemical processes can cause
solute tailing. Physical nonequilibrium is caused by transport
through structure soils, where lateral diffusion of solute in
to and out of the soil matrix or immobile water causes tailing.
Chemical nonequilibrium is caused by sorption/desorption
kinetics. Physical and chemical nonequilibrium processes
can occur simultaneously; however, each soil was repacked(i.e., the structure was disturbed during the column packing
process), which decreased the likelihood of physical nonequilibrium transport. Chloride anion transport experiments
further indicated that physical processes did not cause thesolute tailing. Chloride is a conservative tracer and is normallytransported through soil without sorption. The chloride
breakthrough curves were described with an equilibrium
VOL. 38, NO. 3, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 795

FIGURE 5. Graphical comparison between measured batch isotherm data and Freundlich parameter estimated from the column experiments.
The symbols represent the measured concentrations from the batch experiments at various times. The solid lines represent the isotherms
calculated from the 95% confidence values of the column Freundlich parameter estimates (Table 5). The shaded areas represent possible
isotherms calculable from the combinations of column Kf and n estimates.
advective-dispersive model (i.e., eq 2 with S ) 0) with nosorption, which indicated that the nonequilibrium transport
resulted from chemical interactions (i.e., sorption kinetics).
The nonequilibrium model (eqs 3 and 4) that was used
to describe the breakthrough curves was a fully kinetic
sorption model with degradation/transformation occurring
only on the sorbed phase. In general, the model descriptionsof the column breakthrough curves were excellent (see r2
values, Table 5) and the parameter estimates were reasonable.
In most cases, especially when little or no metabolites were
present (e.g., Gardena and LaDelle), the 95% confidence
interval of the optimized parameters (Table 5) only spanned
realistic values (e.g., the µ values spanned a range thatencompassed values reported for other similar hormones
and were not negative). This small 95% confidence interval
indicates greater confidence in the parameter estimates (29).
The model, eqs 3 and4, did not follow the transport of themetabolites, which may not fully represent some of thecolumns (i.e., Bearden) where metabolites were present. Thismodel nonetheless was reasonable for the columns wherefew, if any, metabolites were present in the effluent. Also, themetabolites that were produced appeared to retain a steroidal
structure, which would have similar physical-chemical
characteristics as testosterone (28) and would likely havevery similar fate and transport parameters as testosterone.
Other models could have been considered and may haveprovided equal or better model fits; however, the number ofparameters would have also increased. Increasing the numberof parameters would have greatly decreased the confidence
in the parameter estimates and in the processes modeled.
796 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 3, 2004

The model that was chosen was simple but included
dominant transport processes that were indicated from thebatch experiments and from other column studies (13) thatused similar hormones (i.e., 17β-estradiol). Furthermore, themodel resulted in excellent descriptions of the measured
data, low 95% confidence intervals of the optimized parameters, and agreement with the batch experiments andprevious studies.
Sorption. The Freundlich sorption parameters that were
derived from modeling the column breakthrough curves
resulted in isotherms that under predicted much of the shorttime (t < 24 h) batch data (Figure 5). The column-derived
isotherms were, however, able to describe some of the latetime batch data. Column experiment values of sorption
usually result in lower sorption estimates than batch sorption
studies because advective transport limits the rate of lateral
diffusion of sorbate to the sorbent. Miscible-displacement
experiments are also more sensitive to isotherm nonlinearity
than batch isotherms (30), which could further decrease theaccuracy at which column derived sorption parameters would
predict batch data. These disparities between batch andcolumn sorption parameters were the reasons why none of
the batch parameters were used as constants in the inverse
column modeling process.
The Kf values reported in Table 5 were more reliable forthe columns where little or no metabolites were present in
the effluent (i.e., Gardena and LaDelle). The 95% confidence
intervals of the Kf values, for the columns where metabolites
were present (i.e., Bearden and Sioux), spanned values thatwere negative. These unrealistic estimates of Kf may reflectthe inability of the model to accurately follow the fate andtransport of the metabolites. Nonetheless, the other parameter estimates (e.g., n, ω, µ) for these columns had 95%
confidence intervals, which provided realistic values.
Testosterone sorption isotherms obtained from soilcolumn experiments were compared to 17β-estradiol (13)and indicated that testosterone had a lower sorption affinity.
It was also found that testosterone was more strongly sorbed
to soil than 17β-estradiol in river and estuary sediments (21).
The lower sorption affinity of testosterone compared to 17βestradiol would explain why field studies (9, 10) have reported
a deeper migration and higher mobility of testosterone
compared to 17β-estradiol.
The long tail of the breakthrough curves in Figure 4
reflected the influence of sorption kinetics (ω value in Table5). This parameter contained the greatest amount of uncertainty compared to the other parameters that were
estimated. Nonetheless, the 95% confidence interval spanned
values that were realistic. The column-derived ω values (Table5) were also only slightly greater than the batch-derived values(Table 3), which further indicated reliable estimates of thisparameter. The column ω values also fell within the rangeof values reported for estrogens in other column (13) andbatch studies (21).
Degradation/Transformations. Parent testosterone waspresent in the column effluent in the current study, unlike
the previous 17β-estradiol studies (13), where no 17β-estradiol
escaped the column. The column µ values (Table 5) weresimilar for all soils, except the Glyndon, where no 14C waspresent in the column effluent. Confidence in the Glyndon
column model results was low because no effluent concentrations could be modeled. The testosterone µ values werecomparable to other values reported by studies done withhormones in agricultural soils and biosolids. Mineralization
rates of testosterone were reported to be 0.912 ( 0.126 h-1
in the presence of biosolids (12), which were twice as large
as values found here. The degradation conditions present inbiosolids may have facilitated higher rates compared to theagricultural soils used in the current study. Furthermore, theoven drying of the soil at 85 °C may have effected the soil
microbial population, which in turn could have decreased
the mineralization rate. Nonetheless, the testosterone µ values
from the current study were almost twice as large as the
17β-estradiol ω values reported for the biosolids (0.252 (0.012 h-1) (12). In agricultural soils, the testosterone µ valuesof the current study were approximately four times greaterthan 17β-estradiol values (0.06-0.12 h-1) reported by Colucci
et al. (32) and fell within the range of values reported by
Casey et al. (13).
The transport of hormones is complicated by simultaneous nonequilibrium transport and transformations. The
experimental results from this study indicated that testosterone sorption affinity was lower than 17β-estradiol (13)and that it transformed more readily. The present study andother field studies (9, 10) have indicated that testosterone
has a greater potential of migration than 17β-estradiol, eventhough it has a higher rate of transformation. These results
underline the importance of the simultaneous transformationand sorption processes in the fate and transport of hormones.
At this time, it is not yet possible to make a definite
statement regarding the potential of testosterone contamination to soil and subsurface water under natural conditions.
In general though testosterone migration through the soil
exists as a potential danger to subsurface water quality as
long as it persists. In one case, testosterone was found to
persist in the soil for 14 days and perhaps longer (10). In thisstudy, it was found that testosterone could escape the upper
soil horizon intact, which means it could potentially migrate
to depths in the soil where biodegradation rates are reduced(i.e., beyond the root zone). If testosterone persists at these

depths, where limited biological activity occurs, then thepotential for subsurface water contamination increases.
These benchtop laboratory experiments provided a deeper
understanding of the fate and transport of testosterone;
however, they used controlled boundary conditions (e.g.,constant temperature, steady-state water flow) and manipulated media (e.g., oven-dried, sieved, repacked soil),which did not represent all natural field conditions. Thesetypes of experimental conditions can be observed in the field
during events such as heavy rainfall or irrigation. Furthermore, the laboratory experiments did not take into consideration various soil heterogeneities (e.g., cracks, root holes,
and worm holes) that can facilitate rapid transport of
testosterone to the groundwater. Further field and laboratory
experiments will be necessary to obtain a definite understanding of hormone fate and transport under natural
conditions.

Acknowledgments
The authors greatly appreciate the long hours dedicated tothis research by the following technical experts: Mrs. Barbara
K. Magelky and Mrs. Colleen M. Pfaff. The authors would
also like to thank the National Science Foundation for theirsupport of this research.

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Received for review July 24, 2003. Revised manuscript received October 16, 2003. Accepted November 12, 2003.
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