Degradation and Sorption of Metribuzin and Primary Metabolites in a Sandy Soil
Trine Henriksen,* Bo Svensmark, and René K. Juhler
ABSTRACT
Leaching to the ground water of metabolites from the herbicide
metribuzin [4-amino-6-(1,1-dimethylethyl)-3-(methylthio)-1,2,4-tria-
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.
zin-5-one] has been measured in a Danish field experiment in concen-
trations exceeding the European Union threshold limit for pesticides
at 0.1 ␮g/L. In the present work, degradation and sorption of metri-
buzin and the metabolites desamino-metribuzin (DA), diketo-metri-
buzin (DK), and desamino-diketo-metribuzin (DADK) were studied
in a Danish sandy loam topsoil and subsoil from the field in question,
using accelerated solvent extraction and liquid chromatography–
tandem mass spectrometry (LC–MS/MS) analysis. Fast dissipation of
metribuzin and the metabolites was observed in the topsoil, with 50%
disappearance within 30 to 40 d. A two-compartment model described
degradation of metribuzin and DA, whereas that of DADK could be
described using first-order kinetics. Part of the dissipation was proba-
bly due to incorporation into soil organic matter. Degradation in
subsoil occurred very slowly, with extrapolated half-lives of more
than one year. Sorption in the topsoil followed the order DA ⬎
metribuzin ⬎ DK ⬎ DADK. Subsoil sorption was considerably lower,
and was hardly measurable for metribuzin and DK. Abiotic degrada-
tion was considerably higher in the topsoil than the subsoil, especially
concerning the de-amination step, indicating that organic matter may
be related to the degradation process. The present results confirm
observations of metribuzin and transformation product leaching made
in the field experiment and demonstrate the need for knowledge on
primary metabolites when assessing the risk for pesticide leaching.
P esticide contamination of ground water and sur-
face water is of increasing concern. In Denmark
pesticides and their metabolites are detected in nearly
half of all monitoring samples (Jørgensen, 2002). The
primary products from metribuzin transformation in soil
are DA, DK, and DADK. Other unidentified metabo-
lites are detected in experiments using 14C-labeled metri-
buzin (Moorman and Harper, 1989; Locke et al., 1994).
Total degradation of metribuzin to inorganic species
(mineralization) is usually below 10% of the metribuzin
applied (Ladlie et al., 1976; Locke et al., 1994; Malla-
watantri et al., 1996) and the highest result measured
is 20% within 90 d (Moorman and Harper, 1989). Thus,
stable metabolites may accumulate in the soil.
In the present study the degradation and sorption of
metribuzin and major metabolites is studied in further
detail. The need for this study of metribuzin degradation
is documented by the recent findings of the metabolites
DK and DADK in high concentrations in soil water and
shallow ground water collected below a field treated
with metribuzin (Kjær et al., 2003). The field (Tylstrup,
T. Henriksen and R.K. Juhler, Geological Survey of Denmark and
Greenland (GEUS), Øster Voldgade 10, DK-1350 Copenhagen, Den-
mark. B. Svensmark, Department of Chemistry, University of Copen-
hagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark. Re-
ceived 8 May 2003. *Corresponding author (the@geus.dk).
Published in J. Environ. Qual. 33:619–627 (2004).
 ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
619
Denmark) is monitored in the Danish Pesticide Leach-
ing Assessment Program. The aim of the program is to
test for leaching at the field scale of pesticide or trans-
formation products resulting from regulatory use (me-
tribuzin dose is 245 g a.i. per 10 000 m2). At a 1- to 6-m
depth, DK was measured in concentrations up to 0.5
␮g/L and DADK up to 2.0 ␮g/L (i.e., 20 times higher
than the maximum allowed level according to the Dan-
ish Environmental Protection Agency). In contrast, me-
tribuzin was detected in very few samples and DA was
absent. Within the past 10 years only four findings of
metribuzin in the ground water exceeded the EU thresh-
old limit for pesticides at 0.1 ␮g/L (Jørgensen, 2002).
This clearly demonstrates that metabolites need to be
considered in ground water monitoring and in pesticide
degradation studies, as pointed out by others (Kolpin
et al., 1998; Lawrence et al., 2001).
Metribuzin degradation is often reported to follow
first-order kinetics (Webster and Reimer, 1976a; Bow-
man, 1991; Locke et al., 1994; Lechon et al., 1997; Di
et al., 1998; Webb and Aylmore, 2002). First-order kinet-
ics is advantageous for use in modeling as a constant
degradation rate and half-life of the pesticide can be
estimated. However, deviations from first-order degra-
dation of metribuzin have been reported. Typically, a
fast initial dissipation is followed by a gradual decrease
in the degradation rate and eventually a very slow long-
term degradation. Consequently, alternative kinetic
models have been used, such as the power–rate equation
developed by Hamaker (1972) employing kinetics hav-
ing an order other than one (Webster and Reimer,
1976b; Hance and Haynes, 1981; Moorman and Harper,
1989) and a first-order function adjusted for decreasing
respiration in the soil samples (Allen and Walker, 1987).
The gradual change in degradation rate may be better
described by using two rate constants instead of one
(Zimdahl et al., 1994; Kjær et al., 2003). The two-com-
partment model (Eq. [1]) describes the degradation pro-
cess as shared between two different compartments,
where degradation proceeds at different rates (k1 and
k2, [d⫺1]). In Eq. [1], C is the concentration (mg/kg) and
t is time (d). The two constants, a and b, express the
quantitative partition between the two compartments,
where a ⫹ b is approximately equal to C0 (mg/kg):
C ⫽ a exp(⫺k1t) ⫹ b exp(⫺k2t) [1]
The fast degradation in the first compartment occurs
when the pesticide is in the soil-water phase and readily
available for microorganisms. In the second compart-
ment the pesticide is sorbed to soil particles. Degrada-
Abbreviations: DA, desamino-metribuzin; DADK, desamino-diketo-
metribuzin; DK, diketo-metribuzin; DT50, dissipation time for 50%
of a pesticide added; DT90, dissipation time for 90% of a pesticide
added; Kd, ratio of sorbed pesticide to pesticide in the aqueous phase;
LC–MS/MS, liquid chromatography–tandem mass spectrometry.
 620 J. ENVIRON. QUAL., VOL. 33, MARCH–APRIL 2004
tion is, therefore, controlled by the rate of desorption–
diffusion into the soil-water phase. The partition be-
tween the two compartments depends on the pesticide
sorption properties and soil characteristics.
In addition to transformation, dissipation of pesticides
in the soil may result from binding to soil material and
irreversible incorporation into soil components or bio-
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.
logical material. The dissipation time for 50% of a pesti-
cide added (DT50) is an important parameter for risk
assessment, but it does not distinguish between the dif-
ferent processes, or account for dissipation time of the
metabolites. Further, only limited data are available con-
cerning metabolite degradation rates, ratio of sorbed
pesticide to pesticide in the aqueous phase (Kd), and
water solubility. In general, degradation changes the
properties of the pesticide toward smaller, more polar,
and often acidic compounds. Thus, metabolites are ex-
pected to be more water-soluble and potentially possess
a higher risk for leaching.
The aim of this study is to provide more detailed
knowledge about the pathway, rate, and mechanism of
degradation for metribuzin as well as the metabolites.
Also, sorption and desorption properties are described.
In the present work degradation and sorption experi-
ments were performed with soil from the Tylstrup field
to benefit from the opportunity to compare the experi-
mental results with field-scale monitoring data.
MATERIALS AND METHODS
Chemicals
Metribuzin (99.5%, CAS RN 21087-64-9) and the degrada-
tion products DA (99%, CAS RN 35045-02-4), DK (98%,
CAS RN 56507-35-0), and DADK (98%, CAS RN 52236-30-3)
were purchased from Dr. Ehrenstorfer (Augsburg, Germany).
Sencor WG (70% metribuzin a.i.) from Bayer A/S (Copen-
hagen, Denmark) was provided by the Danish Institute of
Agricultural Sciences, Flakkebjerg.
Methanol was high performance liquid chromatography
(HPLC) grade from Romil (Cambridge, UK). Water passed
through a Millipore (Billerica, MA) system was used for soil
extraction, sample dilution, and spike solutions, whereas HPLC-
grade water from Rathburn (Walkerburn, Scotland) was used
for the HPLC mobile phase. Acetic acid (pro analysis, 100%)
was purchased from Merck (Darmstadt, Germany). Ottawa
Sand Standard (20–30 mesh) purchased from Fischer Scientific
(Hampton, NH) was used as inert filling material in soil extrac-
tions. R2 Agar was purchased from Difco (Sparks, MD).
Soil
Soil from the Tylstrup field (Kjær et al., 2003) was collected
at two depths, 5 to 20 cm (topsoil) and 3 to 4 m (subsoil, just
above ground water level) to cover the variation in microbial
activity and sorption capacity. Each fraction was thoroughly
mixed, passed through a 5-mm sieve, and stored at 5⬚C in
darkness. Soil properties are shown in Table 1.
Table 1. Soil properties.
Clay Silt Sand
Soil (⬍2 ␮m)† (2–63 ␮m)† (⬎63 ␮m)† Organic C† Water† pH
% air-dried and passed through a 2-mm sieve. Soil (5 g) was trans-
Topsoil 4.16 20.38 75.16 1.47 15.4 5.50
Subsoil 1.14 21.52 77.35 0.056 22.4 5.98
† Percentage of dry weight.
Part of the soil was sterilized to study abiotic degradation
at the different steps in the degradation pathway. Packages
of soil in airtight bags were treated with 2 ⫻ 25 kGy ␥-radiation
(LR Plast, Glostrup, Denmark). The bags were stored in dark-
ness at 5⬚C and kept closed until use.
Sample Preparation for Degradation Experiments
The herbicide formulation Sencor WG was used for the
degradation experiments to imitate field degradation. Analyti-
cal-grade metribuzin was added to a small number of samples
for comparison. Degradation of the metabolites DA, DK, and
DADK was studied independently in the four types of soil
using the metabolite as spike compound. The initial concentra-
tion of the spike compound in the soil samples was about 0.5
mg/kg. This corresponds to the level in the upper 10 cm of
the soil if the maximum dose of metribuzin is sprayed in one
treatment. Even if metabolite concentration in the subsoil may
be lower, a uniform level was chosen for all samples and
compounds to evaluate a worst-case scenario. The initial con-
centrations in the soil samples were as follows: metribuzin
(Sencor) ⫽ 0.53 mg/kg; metribuzin (analytical standard) ⫽
0.43 mg/kg; DA ⫽ 0.67 and 0.62 mg/kg for topsoil and subsoil,
respectively; DK ⫽ 0.46 and 0.30 mg/kg for topsoil and subsoil,
respectively; and DADK ⫽ 0.67 mg/kg.
Soil corresponding to 25 g dry wt. was transferred to a 250-mL
blue cap flask. Aqueous spike solution (1 mL) was added to
the soil surface as evenly as possible. For the sterile samples,
the flasks were autoclaved before use and weighing and spiking
was performed in a flow-bench with sterilized tools. Finally,
the flasks were incubated at 10 ⫾ 1⬚C, simulating the average
Danish soil temperature at 1 m below the soil surface. Once
a month the flasks were opened for 10 min to maintain aero-
bic conditions.
Sterile spike solutions were prepared to prevent contamina-
tion of the sterile samples. A small amount of solid compound
was dissolved directly in 2 or 5 mL of autoclaved MilliQ water
(Millipore). This solution was filtered through a 0.2-␮m sterile
nylon filter into a blue cap flask containing the remaining
volume of autoclaved water. The exact concentrations were de-
termined afterward by liquid chromatography–tandem mass
spectrometry (LC–MS/MS). To dissolve the metabolites DK,
DA, and DADK, 250 ␮L of acetone was added to the primary
solutions. The final concentration of acetone in the spike solu-
tion was 0.5% (v/v), which was not considered to influence
the soil bacteria.
After 7 mo a number of sterile soil samples and two nonster-
ile samples were tested for microbial activity. From each sam-
ple 10 to 15 g soil was thoroughly shaken with 100 mL auto-
claved 0.9% NaCl solution. An aliquot of the aqueous phase
(100 ␮L) was spread on R2 Agar plates and incubated for
90 d at 20⬚C. No bacterial growth was observed at the plates
derived from the sterile soil samples within this period.
Sample Preparation for Sorption Experiments
Sorption of metribuzin, DA, DK, and DADK (in mixtures
and separately) was measured by batch experiments. Mixture
samples were prepared with initial concentrations of each
compound at 50, 75, 100, 150, and 250 ␮g/L in the aqueous phase,
except for DK being at 75 to 375 ␮g/L. In addition, control
samples of each compound alone were prepared at the highest
concentration, 250 ␮g/L. Soil for the sorption experiments was
ferred to a 12-mL screw cap test tube. Four milliliters of
“artificial ground water” were added, made up from tap water
and MilliQ water in equal proportions to obtain an ion strength
 HENRIKSEN ET AL.: DEGRADATION AND SORPTION OF METRIBUZIN
similar to average Danish ground water. After equilibration
for 24 h, 1-mL standard solution was added to obtain the
respective concentrations. The samples were equilibrated at
10⬚C in the dark for 96 h. Before analysis, the samples were
centrifuged for 30 min at 1500 ⫻ g and an aliquot of the
supernatant solution was filtered through a 0.20-␮m filter and
analyzed by LC–MS/MS.
Desorption experiments were performed using topsoil sam-
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.
ples. The supernatant was removed and the remaining water
volume was calculated by weighing. For desorption experi-
ments 4 mL of fresh “ground water” was added to each sample.
The samples were equilibrated for 96 h at 10⬚C and analyzed
as the sorption experiments.
Soil Extraction
Extraction and analysis followed the method described in
Henriksen et al. (2002) with minor adjustments. Soil samples
were extracted with methanol and water (75:25 v/v), using an
Accelerated Solvent Extraction 200 from Dionex (Sunnyvale,
CA). The samples were equilibrated with the solvent for 10
min at 60⬚C and 10.3 MPa, followed by flushing with fresh
solvent and purging with nitrogen.
An aliquot of the soil extract was filtered through a 0.45-␮m
nylon filter. This aliquot was further diluted three to six times
with methanol and water (75:25, v/v) to obtain a concentration
of the original compound of about 50 ␮g/L. The undiluted
extract was used for analysis of the metabolites produced in
the samples.
Liquid Chromatography–Tandem Mass
Spectrometry Analysis
Analysis was performed with a Waters (Milford, MA) 2690
high performance liquid chromatography system, connected to
a Quattro Ultima triple-quadrupole mass spectrometer from
Micromass (Manchester, UK). Electrospray ionization (ESI)
was used in the positive mode for metribuzin and DA, and
in the negative mode for DK and DADK. Analytes were
separated with an XTerra RP18 column from Waters (2.1 ⫻
100 mm, 3.5-␮m particle size) using a mobile phase of metha-
nol and 0.1% acetic acid (50:50, v/v) with flow rate at 180 ␮L/
min. Capillary voltage was 2.80 to 3.20 kV, cone voltage 50
to 80 V, and collision energy for tandem mass spectrometry
18 to 20 eV, depending on the analyte. The injected sample
volume was 10 ␮L. Analytes were quantified by selected reac-
tion monitoring, measuring a single characteristic fragment
ion of each analyte. The respective ion traces were as follows:
metribuzin, m/z 215 → 187; DA, m/z 200 → 172; DK, m/z
183 → 139; and DADK, m/z 169 → 97.
Data Analysis
In each series of sample analysis the content of metribuzin
and metabolites in the soil samples was adjusted for variations
in extraction efficiency by including freshly spiked recovery
control samples. Despite this procedure, a rather high day-
to-day variation of recovery was observed in the subsoil sam-
ples, which exceeded the variation among the triplicates. In
the topsoil the random day-to-day variation was much less pro-
nounced.
Curve fittings for degradation kinetics and Freundlich iso-
therms were performed in SigmaPlot (SPSS, 1999). For fittings
to the two-compartment model, constraints for acceptable fits
were chosen for the variables a and b (Eq. [1]) to be within
0 and 130, as the ideal sum of a ⫹ b is 100(%).
To quantify how sorption of the individual compound was
influenced by the other compounds present in the mix samples,
621
a ratio was calculated for the control samples and the mix
samples (Kd,mix/Kd,control) having equal initial concentrations of
about 250 ␮g/L. Differences between metabolite formation in
nonsterile and sterile soil were tested by two-way ANOVA
for repeated measurements performed in Excel 5.0 (Micro-
soft, 2003).
RESULTS AND DISCUSSION
The aim of this study was to study the degradation
process of metribuzin and the main metabolites in top-
soil and subsoil. Furthermore, to compare biotic and
abiotic degradation, sterile soil samples were included
in the study. Despite the fact that soil sterilization by
irradiation is reported to cause the smallest change in
soil characteristics (Stroetmann et al., 1994), more or-
ganic material was released to the soil extracts from the
sterile topsoil than from the nonsterile soil, indicating
some destruction of the soil structure. Also, higher re-
coveries of the compounds were obtained in the sterile
soil (exceeding 100% of the amount applied in the first
measurements), which must be due to differences in
binding compared with the nonsterile soil, which was
used for the recovery control samples. Finally, detection
and quantification of DK in the topsoil was difficult
due to interference from co-eluting matrix substances,
especially in the sterile topsoil, which is why data in
some cases are lacking or excluded due to inadequate
quantification.
Metribuzin Degradation
In the topsoil, fast initial dissipation of metribuzin
was observed, with less than 50% of the applied amount
of metribuzin remaining after 30 d. After this, the rate
gradually decreased and 5% could be recovered one
year after incubation. When comparing experiments us-
ing the formulation Sencor WG with the pure analytical
standard, no difference in metribuzin dissipation could
be observed. The experimental data were better de-
scribed using the two-compartment model rather than
the first-order model. The two-compartment fit is shown
in Fig. 1a. However, a possible lag phase was not de-
scribed by the two-compartment model, resulting in an
overestimate of the constants a and b (Eq. [1]; a ⫹ b
should be close to C0, corresponding to 100%). The
best-fit equations for degradation of metribuzin and the
metabolites, as well as dissipation time for 50 and 90%
of a pesticide added (DT50 and DT90, respectively) calcu-
lated using these equations, are listed in Table 2.
Previous studies of metribuzin degradation in the top-
soil at field or laboratory conditions have reported DT50
values in the range 11 to 46 d (first-order kinetic, 20–
25⬚C) (Hyzak and Zimdahl, 1974; Bowman, 1991; Gal-
laher and Mueller, 1996; Lechon et al., 1997; Di et al.,
1998). However, high values of 75 d (Sharom and Ste-
phenson, 1976) and 145 d (Webb and Aylmore, 2002)
have been measured. Compared with those values (ex-
cept from the last two), dissipation of metribuzin in the
present study appeared to be slower, considering the DT90
of 149 d. However, it has been shown that the degrada-
tion of metribuzin is highly temperature-dependent.
 622 J. ENVIRON. QUAL., VOL. 33, MARCH–APRIL 2004

Table 2. Kinetic equations and dissipation time for 50 and 90%

of a pesticide added (DT50 and DT90, respectively) for metri-
buzin and metabolites in topsoil and subsoil,
First-order or
Chemical† two-compartment model‡ r DT50 /DT90
d
Topsoil
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.

Metribuzin y ⫽ 130 exp(⫺0.057x ) ⫹ 0.990 32/149

38.2 exp(⫺0.009x )
DA y ⫽ 130 exp(⫺0.060x ) ⫹ 0.997 29/97
40.7 exp(⫺0.015x )
DK did not fit any model – –
DADK y ⫽ 131.1 exp(⫺0.020x ) 0.992 35/129
Subsoil
Metribuzin y ⫽ 115.5 exp(⫺0.001x ) 0.571 ⬎500
DA y ⫽ 12.8 exp(⫺0.048x ) ⫹ 0.796 ⬎500
93.9 exp(⫺0.0007x )
DK y ⫽ 38.9 exp(⫺0.028x ) ⫹ 0.906 ⬎500
90.3 exp(⫺0.0004x )
DADK y ⫽ 37.9 exp(⫺0.011x ) ⫹ 0.822 ⬎500
73.1 exp(⫺0.0001x )
† DA, desamino-metribuzin; DADK, desamino-diketo-metribuzin; DK,
diketo-metribuzin.
‡ First-order equation was used if the two-compartment model did not
improve correlation.

porated, as it could not be released by the rather harsh

Fig. 1. Dissipation of metribuzin in (a) the topsoil and formation
of the metabolites desamino-metribuzin (DA), diketo-metribuzin
(DK), and desamino-diketo-metribuzin (DADK) in (b) the non-
sterile and (c) the sterile topsoil. Vertical bars in (a) represent the
standard deviation of metribuzin in the triplicate samples. The
amount of metabolite formation in (b) and (c) is calculated as
percent of metribuzin applied on a molar basis. The metabolite DK
was not detected in the sterile topsoil due to matrix interference.
Relative standard deviations for the metabolites were on average
39% (DK), 6% (DA), and 8% (DADK).
Half-lives increase 6 to 11 times when lowering the tem-
perature from 20 to 25⬚C to 5⬚C (Hyzak and Zimdahl,
1974; Lechon et al., 1997). Thus, the low incubation
temperature of 10⬚C used in the present study may ex-
plain the slower dissipation observed.
In Fig. 1, the topsoil dissipation of metribuzin and
formation of metabolites are shown for the sterile and
nonsterile soil. It is notable that the steep decrease in
metribuzin concentration is similar for both curves in
Fig. 1a. In the period from Days 16 to 33, the content
of metribuzin decreased from 90 to 40% of the initial
amount in the nonsterile soil and from 120 to 80% in
the sterile soil (Fig. 1a). Within the same time the forma-
tion of DA, DK, and DADK in the nonsterile topsoil
(Fig. 1b) corresponded to only 12% of the initial amount
of metribuzin, calculated on a molar basis, and only 3%
in the sterile soil (Fig. 1c). Thus, comparing the fast
dissipation of metribuzin within this period with the
formation of metabolites in the nonsterile and sterile
soil, it is clear that the dissipation cannot result from
degradation only. Even if other unknown metabolites
might have been formed from DADK within this time,
this is unlikely to explain the total loss. It is more likely
that part of the decrease in metribuzin is caused by
strong sorption to the soil organic matter, possibly incor-
extraction conditions applied. During the following time
interval (Days 33–137, Fig. 1a and 1b), better agreement
was observed between metabolite formation and metri-
buzin dissipation in the nonsterile soil, indicating that
degradation had become the dominating process. Even
if only 5% metribuzin was remaining after one year of
incubation, degradation and release of metabolites may
still occur. On a long time scale the formation of bound
metribuzin residues may not be a totally irreversible pro-
cess. For instance, metribuzin that is covalently bonded
to humic acids can be released as de-aminated metri-
buzin by hydrolysis as demonstrated by Landgraf et al.
(2001). Also, slow diffusion into micropores and organic
matter may be reversed if the gradient changes. Thus,
metabolites formed at the end of the experiment may
derive from metribuzin released from the soil by desorp-
tion. At the Tylstrup field, leaching of DK and (primary)
DADK from the root zone was still observed three years
after metribuzin application. This clearly indicates the
presence of a slow long-term desorption and degrada-
tion (Kjær et al., 2003).
Subsoil degradation of metribuzin occurred very
slowly, as illustrated in Fig. 2. About 80% of the applied
metribuzin was still present after one year. This is consis-
tent with the formation of metabolites, predominantly
DA and DK, which reached a level of 10% of applied
metribuzin. The data did not fit very well to any kinetic
equation. Using a first-order fit, a half-life exceeding
500 d was calculated. There was no indication of strong
sorption of metribuzin in the subsoil or systematic differ-
ences between sterile and nonsterile degradation.
It is a common observation that degradation rates
below the root zone are significantly slower, primary
due to lower microbial activity. Reported half-lives for
metribuzin range from 50 to 222 d (Moorman and Har-
per, 1989; Di et al., 1998; Webb and Aylmore, 2002).
These values are notably shorter than in the present study
 HENRIKSEN ET AL.: DEGRADATION AND SORPTION OF METRIBUZIN
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.
Fig. 2. Dissipation of metribuzin in (a) the subsoil and formation of
metabolites desamino-metribuzin (DA), diketo-metribuzin (DK),
and desamino-diketo-metribuzin (DADK) in (b) the nonsterile
subsoil and (c) the sterile subsoil. Vertical bars in (a) represent
the standard deviation of metribuzin in the triplicate samples. The
amount of metabolite formation in (b) and (c) is calculated as
percent of metribuzin applied on a molar basis. Relative standard
deviations for the metabolites were on average 31% (DK), 7%
(DA), and 17% (DADK).
and may be due to different experimental conditions with
respect to soil depth and incubation temperature.
Metabolite Formation
In the nonsterile topsoil, the metabolite DA reached
a level of 6 to 7% of applied metribuzin within a short
time interval after spiking with metribuzin (Fig. 1b).
The DA formation was immediately followed by forma-
tion of DADK. A high peak of DK was observed at
Days 60 to 80 apparently followed by a further increase
of DADK, indicating a rapid transformation of DK as
well as DA. Even if a relatively high content of DK was
measured at Day 375, the rather constant presence of
DA during the whole incubation period, concurrent with
the fast disappearance of DK, indicate that the slow
long-term degradation may occur primary via the DA
pathway. Also in the sterile topsoil, DA was found dur-
ing the whole incubation period, but in a lower concen-
tration (Fig. 1c), and especially the subsequent forma-
tion of DADK was notably lower. Matrix interference
in the sterile topsoil was too high to detect DK at a
level realistic for metabolite formation. Thus, no results
for DK are presented in Fig. 1c even though DK may
have been present in the samples at levels below 0.03
mg/kg (approximately 10% of applied metribuzin).
Formation of DA in soil may occur by photochemical
623
reactions at the soil surface and by biotic and abiotic
processes in the soil (Hatzios and Penner, 1988; Schilling
et al., 1985; Webster and Reimer, 1976b). Recently, a
mechanism was suggested by Landgraf et al. (2001) for
abiotic degradation of metribuzin to DA by interaction
with the humic substances in the soil. Using infrared
spectroscopy the changes in chemical bonding and func-
tional groups were studied by adding metribuzin to hu-
mic acid. An increase in amide bonds and a decrease
in alcohols were observed. They suggested reactions
between the metribuzin amino group and carboxylic
acid and alcoholic functional groups in the humic sub-
stances, followed by the release of DA. The steady for-
mation of DA in the sterile topsoil in the present work
supports this theory.
Degradation of Metabolites
Topsoil and subsoil degradation of each metabolite
are shown in Fig. 3. In the topsoil only 8 to 16% of the
respective metabolites applied were remaining after 125 d.
Similar to metribuzin, the loss was probably partly
caused by other processes than degradation as shown
by results for the sterile soil samples.
In the sterile topsoil, only about 50% DA was left
after 58 d (Fig. 3b) even though the formation of DADK
in these samples did not exceed 4% of applied DA
(molar basis). A similar decrease was observed in the
samples spiked with DADK (Fig. 3c), but as the analysis
did not include any subsequent metabolites it is not
clear whether this loss of DADK was due to incorpora-
tion, strong sorption, or abiotic degradation. However,
some deductions can be made from the best-fit equa-
tions. Degradation of DADK (in the nonsterile soil)
was better described by first-order kinetics than by the
two-compartment model. The opposite was observed for
DA degradation, indicating that degradation of DADK
was less influenced by sorption–desorption than DA.
Diketo-metribuzin dissipated rather fast in both the
sterile and nonsterile topsoil, and it is notable that com-
parable levels were reached in the two soils after 90 d
(Fig. 3a). However, variation in the content of DK in
the nonsterile soil between the first measurements
makes the results less decisive. Considering the few data
points in the DK curves, degradation of DK can also
be evaluated indirectly by considering formation of the
metabolite DADK in these samples. In the nonsterile
samples, DADK reached a level of 14% of applied DK
within 14 d, and it was still 9% in the last measurement,
indicating a steady degradation of DK. The level of
DADK was almost the same in the sterile samples, and
at the end of the experiment it exceeded the nonsterile
samples (Fig. 3a). The relatively high degree of abiotic
degradation of DK compared with DA (Fig. 3b) may
be due to the same reaction with the humic substances
as described for metribuzin (Landgraf et al., 2001). The
amino group is still present in DK and the mechanism
causing de-amination may be similar. Differing from the
present results, DK was found in a field experiment to
be the most persistent of the three metabolites and the
only metabolite present in the soil the following season
 624 J. ENVIRON. QUAL., VOL. 33, MARCH–APRIL 2004
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.

Fig. 3. Dissipation and transformation of the metabolites in the sterile and nonsterile topsoil and subsoil. Topsoil: (a) diketo-metribuzin (DK),
(b) desamino-metribuzin (DA), and (c) desamino-diketo-metribuzin (DADK). Subsoil: (d) DK, (e) DA, and (f) DADK. Vertical bars represent
the standard deviation in the triplicate samples. Formation of DADK from DK (a and d) and DA (b and d) is calculated as percent of the
applied compound on a molar basis.

after spraying (Webster and Reimer, 1976a). Subsoil
degradation was very slow for all the metabolites (Fig.
3d–f) and fitting such data to a model is delicate as the
dissipation time calculated exceeds the incubation time.
Taking this into consideration the two-compartment fit
demonstrated the best correlation. The DT50 values cal-
culated were 900 d or more (Table 2).
Abiotic Degradation
The importance of microbial and abiotic transforma-
tion in the steps of the degradation pathway was studied
by comparing the primary metabolite formation from
metribuzin (DA and DK) in the sterile and nonsterile
soil samples, and by comparing formation of DADK
from the samples treated with DA and DK, respectively.
Significant difference (p ⬍ 0.05) was found in all cases
by two-way analysis of variance (ANOVA), both in the
topsoil and the subsoil. Further, to estimate the relative
contribution from biotic and abiotic degradation the
quantitative differences were calculated. For example,
in the samples originally treated with DA, the microbial
degradation is estimated by subtracting the content of
DADK (the triplicate means) in the sterile soil samples
from the content of DADK in the nonsterile soil, at
each point of measurement. This is illustrated in Fig. 4,
showing the difference curves for the formation of
DADK in nonsterile and sterile topsoil by transforma-
tion of DA and DK, respectively. It was assumed that
the more positive the difference, the more dominating
was the microbial degradation. However, this can only
be a rough estimate since metabolite accumulation is
the result of metabolite formation and metabolite degra-
dation processes. As seen in Fig. 4, the difference de-
creases over time, probably do to a higher degree of
metabolite accumulation in the sterile soil, which is why
estimation of biotic and abiotic degradation should pri-
marily be based on the first part of the curves. Accordant
to that, it appears in Fig. 4 that the difference between
 HENRIKSEN ET AL.: DEGRADATION AND SORPTION OF METRIBUZIN 625
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.

Fig. 4. Difference between formation of desamino-diketo-metribuzin

(DADK) in nonsterile and sterile topsoil via desamino-metribuzin
(DA) and diketo-metribuzin (DK). The vertical bars represent the
standard deviation for the calculated differences using the rule for
propagation of errors.

biotic and abiotic degradation is bigger for the step

DA → DADK than for DK → DADK, indicating that
the transformation of DA is more microbially depen-
dent than that of DK.
By similar calculations and considerations, it was
found that the DA pathway in the topsoil had a consider-
able contribution from abiotic degradation in the first step
MBZ → DA, whereas the second step DA → DADK
was predominantly microbially dependent as illustrated
in Fig. 4. In the DK pathway in the topsoil, the first
step MBZ → DK could not be evaluated, since DK was
not detected in the sterile topsoil spiked with metri-
buzin. The second step, de-amination of DK to form
DADK, had a considerable contribution from abiotic
degradation (Fig. 4).
In the subsoil, the differences were considerably smaller
due to the low degree of degradation in general. The
most distinct difference was seen for the step DK →
DADK, which had a relatively high contribution from
microbial degradation compared with abiotic degrada-
tion. Overall, more degradation took place in the sterile
topsoil than in subsoil (sterile as well as nonsterile). In
particular the de-amination process proceeded much
faster in the topsoil, supporting the hypothesis that
humic substances are involved in the process of abi-
otic degradation.

Sorption and Desorption

Freundlich isotherms were determined for sorption
and desorption of the four compounds in the topsoil
(Fig. 5). In the subsoil isotherms could be determined
for DA and DADK only, since the subsoil sorption of
metribuzin and DK was offset by the variation within
the triplicates. The Freundlich constants for the sorption
and desorption isotherms are summarized in Table 3.
The sorption isotherms indicated saturation (n ⬍ 1)
in all cases except for a linear isotherm of DADK in
the topsoil.
Under field conditions, metribuzin and one or several
Fig. 5. Freundlich isotherms for sorption and desorption of metri-
buzin and the metabolites in the topsoil. Vertical bars represent
the standard deviation on sorbed pesticide calculated from the
deviation on pesticide in the aqueous phase. The results are pro-
duced from mixed samples, containing all four compounds.
metabolites are likely to be present in the soil simultane-
ously. To study possible interactions, the isotherms in
this work are based on measurements using samples
containing all the four compounds in mixture in equal
concentrations. Thus, interaction between compounds
 626 J. ENVIRON. QUAL., VOL. 33, MARCH–APRIL 2004

Table 3. Freundlich isotherms and Kd,mix/Kd,control for sorption and (Bowman, 1991). In contrast, DK was found to be the
desorption. strongest sorbed species in a batch experiments with
Freundlich isotherm
Kd,mix/
Dundee loam (fine-silty, mixed, active, thermic Typic
Chemical† Kf (⫾SD) n (⫾SD) R Kd,control‡ Endoaqualfs) (Locke et al., 1994). This may result from
Topsoil sorption
the presence of different types of sorption sites in the
Metribuzin 3.33 ⫾ 2.46 0.73 ⫾ 0.17 0.941 0.90
more clayey soil. Also, the soil pH of the Dundee loam
DA 8.67 ⫾ 3.68 0.59 ⫾ 0.11 0.959 0.75 soil was higher (6.43). In another study on metribuzin,
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.

DK 7.25 ⫾ 1.99 0.53 ⫾ 0.05 0.986 0.74 a negative correlation between sorption and soil pH was
DADK 0.38 ⫾ 0.14 1.01 ⫾ 0.08 0.993 0.55
demonstrated. At pH 6.7 the sorption was about twice
Topsoil desorption
as the sorption at pH 4.6 (Ladlie et al., 1976). However,
Metribuzin 1.49 ⫾ 1.69 1.23 ⫾ 0.34 0.919 1.03
DA 35.3 ⫾ 11.9 0.75 ⫾ 0.28 0.912 1.77
the sensitivity to pH may not be equal for metribuzin
DK 2.89 ⫾ 0.30 0.93 ⫾ 0.03 0.998 0.22 and the metabolites. Consequently, variations in soil pH
DADK 4.50 ⫾ 0.09 0.25 ⫾ 0.01 0.999 0.06 may result in a change of the mutual order of sorption.
Subsoil sorption In the subsoil the sorption of DA and DADK was
DA 0.56 ⫾ 0.17 0.89 ⫾ 0.07 0.995 0.32 lower and isotherms for metribuzin and DK could not
DADK 0.21 ⫾ 0.35 0.91 ⫾ 0.33 0.914 0.24
be calculated. This may be due to a much lower sorption
† DA, desamino-metribuzin; DADK, desamino-diketo-metribuzin; DK, capacity in the subsoil. Considering DA and DADK, it
diketo-metribuzin.
‡ Kd ⫽ Cs /Cw measured in the control sample and the mix sample of the is notable that the order of sorption differs from the
highest concentration. The term Kd is the ratio of sorbed pesticide (Cs) topsoil, with strongest sorption in the subsoil of the
to pesticide in the aqueous phase (Cw). two de-aminated metabolites DA and DADK. A likely
cause is that other mechanisms may be involved in the
and competition for active sites on the soil components subsoil sorption in which the amino group affects the
may occur in these samples. For each single compound equilibrium toward the solution phase.
separate control samples were prepared at the highest Desorption was studied in the topsoil only, due to the
concentration. The effect of the other compounds pres- low sorption in subsoil. As seen in Fig. 5, the desorption
ent in the sample is expressed by the ratio Kd,mix/Kd,control isotherms are steeper than the sorption isotherms and
(likewise for Kdes, the equilibrium constant for the de- displaced to the left, indicating hysteresis for metribuzin,
sorption process, i.e., ratio of sorbed pesticide to pesti- DA, and DK (except for the last data point for DK,
cide in the aqueous phase). In most cases a lowering of see below). Interestingly, the desorption isotherm for
sorption in the mix samples (ratio of ⬍1) is observed DADK is less steep, with the last two data points drop-
(Table 3). This mixture effect has to be considered when ping off toward zero (not included in the isotherm).
comparing the values in Table 3 with values published This is probably caused by strong interactions among
by others, obtained from separate measurements. Even the compounds in the mix samples during the desorption
if less sorption of the individual compounds was ob- process. Comparing Kdes at the highest concentration
served in the mix samples, the sum of sorbed fractions for the mix samples and the separate control samples,
was higher in the mix than in the individually spiked significant divergences were observed except for metri-
samples. This may result from the presence of different buzin. It appears from the ratios in Table 3 that Kdes for
sorption sites having dissimilar specificity toward com- DA was almost twice as high in the mix samples. The
pounds. In principle, the mix sample sorption might be opposite was observed for DK and DADK. Also, hyster-
mistaken for degradation and thereby cause over- or esis was observed for DADK in the separate samples
underestimation of sorption coefficients. However, this only. In conclusion, the process of desorption appears to
is not feasible as no degradation was observed in the be more influenced by the total concentration of com-
single-compound samples. pounds in solution than the sorption process. The mech-
Considering sorption of organic pollutants the soil anism might be that in the mix samples DA gradually
content of organic matter is often considered of highest takes over the unoccupied sites left by desorbing DK
importance. For metribuzin, Kd varied from 31.7 L/kg and DADK. Consequently, the equilibrium continu-
in a soil containing 60% organic matter to 0.56 L/kg in ously displaces in favor of more DA sorbed to the soil
a very sandy loam (Sharom and Stephenson, 1976). In in exchange for DADK and DK.
accordance, in the present study on metribuzin a Kd of This type of measurement may be more descriptive
0.94 L/kg was measured in the low organic sandy loam of the conditions relevant for field conditions. Hence,
(in the topsoil control sample). during metribuzin transformation in the environment it
Considering topsoil, the metabolite DADK had the can be anticipated that the active compound as well as
lowest sorption followed by DK (comparing calculated transformation products are present simultaneously in
Kd values at equal concentration). This is in agreement the soil. This aspect should be considered when em-
with the findings resulting from field applications of the ploying results from single-compound experiments.
pesticide as these two transformation products were
monitored in the ground water and pore water at the
Tylstrup field (Kjær et al., 2003). Also, similar results CONCLUSIONS
were seen in a lysimeter experiment where the mobility Metribuzin was degraded in the topsoil with the for-
followed the order DADK ⬎ DK ⬎ metribuzin ⬎ DA mation of all the three metabolites, DA, DK, and DADK.
in Plainfield sand (mixed, mesic Typic Udipsamments) Initial dissipation was relatively fast, followed by a de-
 HENRIKSEN ET AL.: DEGRADATION AND SORPTION OF METRIBUZIN
crease in the rate. Metribuzin and DA kinetics were
best described using a two-compartment model, and
sorption as well as fast in-solution degradation contrib-
uted to the fast initial dissipation. Dissipation of DADK
could be best described using simple first-order kinetics
whereas data for DK did not fit any kinetic equation.
However, the degree of DK degradation seemed compa-
Reproduced from Journal of Environmental Quality. Published by ASA, CSSA, and SSSA. All copyrights reserved.
rable with DA. In the subsoil degradation occurred very
slowly for all the compounds, with DT50 estimated at
more than 500 d.
Abiotic degradation of metribuzin and the metabo-
lites was notably faster in the topsoil than in the subsoil,
implying that the humic substances in the soil are di-
rectly involved in or facilitate transformation, especially
the de-amination process.
Sorption in the topsoil followed the order DA ⬎
metribuzin ⬎ DK ⬎ DADK. Desorption followed the
same order, but the differences were more pronounced.
Important differences between measurements made in
single-component and mixture experiments were ob-
served. When present in mixture, the metabolite DA
was desorbed to a less extent at the expense of DK
and DADK and an exchange mechanism was suggested.
Sorption to the subsoil was lower, and only measurable
for the de-aminated metabolites DA and DADK.
From the present study, it may be expected that the
majority of metribuzin will dissipate during the growth
season due to degradation, strong sorption, and bound
residue formation. Both metabolites DA and DK are
formed in the degradation process followed by DADK.
Slow degradation continues for a long period, primarily
controlled by the desorption process. Due to differences
in sorption and especially desorption properties, metri-
buzin and DA will preferentially be retarded in the
topsoil whereas DK and DADK are considerably more
present in the solution phase and thus exposed to leach-
ing. As DK and DADK move through the subsoil layers,
DADK will be most retarded, causing DK to reach the
ground water in a higher extent and before DADK.
This scenario is in good agreement with monitoring
results from a field-scale monitoring program. Also, the
present work demonstrates the need for knowledge of
the primary metabolites with respect to degradation,
sorption, and desorption properties when assessing the
risk of pesticide leaching, and the multicompound ef-
fects on such processes are documented.
ACKNOWLEDGMENTS
Technical assistance and advice from L. Gudmundsson, M.
Andersen, P. Stockmarr, P.B. Jacobsen, and M. Schou (GEUS)
are gratefully acknowledged. Thanks to J. Kjær for valuable
discussions regarding the Tylstrup monitoring results.
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