Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue5.2000:
Soil Biology & Biochemistry 32 (2000) 615±625
www.elsevier.com/locate/soilbio
Dependence of accelerated degradation of atrazine on soil pH in
French and Canadian soils
Sabine Houot a,*, Edward Topp b, Abdellah Yassir c, 1, Guy Soulas c
a
I.N.R.A. Unite de Science du Sol, BP 01, 78850 Thiverval-Grignon, France
Pest Management Research Centre, Agriculture and Agrifood Canada, 1391 Sandford Street, London, Ontario, Canada N5V 4T3
c
I.N.R.A.-C.M.S.E., Laboratoire de Microbiologie des Sols, 17 rue Sully, BV 1540, 21034 Dijon Cedex, France
b
Accepted 30 September 1999
Abstract
A series of agricultural soils varying in their atrazine treatment history were sampled from 12 sites in France and two sites in
Canada. The soils varied widely with respect to soil chemical, physical and microbiological (total microbial biomass, kinetics of
C and N mineralization) properties. Soils treated with as few as two successive atrazine ®eld applications mineralized [Uring-14C]atrazine signi®cantly more rapidly in 35 d laboratory incubations than did soils which had never received atrazine.
Longer treatment history tended to favour more rapid mineralization in the so-called ``adapted'' soils. Up to 80% of the initially
applied 14C-atrazine was mineralized at the end of the incubations in these adapted soils. Of the properties tested, soil pH was
the most signi®cantly related to atrazine mineralized. In soils with pH lower than 6.5, less than 25% of the initial 14C-atrazine
was mineralized even after repeated application in ®eld conditions. Atrazine retention in soil did not in¯uence its mineralization
rate. Both hydroxylated and dealkylated atrazine metabolites were detected, but no clear pattern of metabolite production could
be determined. Large amounts of bound residues were formed in soils that mineralized little atrazine. 7 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Atrazine; Accelerated mineralization; Soil; pH
1. Introduction
Repeated application of some xenobiotic pesticides
to soil can result in the adaptation and development of
a soil micro¯ora which can rapidly metabolize the
compound (Racke and Coats, 1990). In some cases
accelerated degradation can result in loss of ecacy
and signi®cant economic cost (Suett et al., 1996a).
Accelerated degradation of a number of herbicides,
including phenoxyalkanoic acids, thiocarbamates
(Roeth, 1986) linuron, propyzamide, metamitrone and
* Corresponding author. Tel.: +33-1-3081-5401; fax: +33-1-30815396.
E-mail address: [email protected] (S. Houot).
1
Present address: Universite Caddi-Ayyad, Faculte des Sciences
Semlalia, DeÂpartement de Biologie, BPS 15, Marrakech, Maroc.
napropamide (Walker and Welch, 1992) has been
documented. Atrazine [6-chloro-N2-ethyl-N4-isopropyl-1.3.5-triazine-2.4-diamine] is a herbicide widely
used in maize production. It was considered relatively
recalcitrant in soils, although microbial degradation
has always been recognized as the principal mechanism
of atrazine dissipation in soils (Kaufman and Kearney,
1970). Until recently, N-dealkylation reactions catalyzed by bacteria or fungi was the classical pathway of
microbial degradation described (Kaufman and Kearney, 1970; Mougin et al., 1994; Nagy et al., 1995).
Complete and rapid mineralization of the 14C-labelled
s-triazine ring, both by soil populations and by bacterial isolates, is now commonly observed (Assaf and
Turco, 1994; Yanze-Kontchou and Gschwind, 1994;
Mandelbaum et al., 1995; Radosevitch et al., 1995;
Barriuso and Houot, 1996; Vanderheyden et al., 1997).
The ring-cleavage substrate is cyanuric acid, which is
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 8 8 - 1
616
Table 1
Location, cropping and management, and primary physical and chemical properties (expressed on a dry weight basis) of the soils used in this studya
Location
Management and crop in 1996
Particle size (%)
clay
silt
Canadian soils
Plots frequently receiving atrazine (continuous maize or rotation including maize)
Harrow 1
R
M
47.3
Harrow 2
R
M
idc
Harrow 3
C
M
id
24.2
id
id
C
(g kgÿ1)
30.9
5.7
41.4
43.0
5.8
48.0
12.6
15.0
35.6
17.6
14.8
15.3
14.7
10.6
4.3
4.8
4.5
4.1
4.3
4.1
4.1
5.2
5.1
5.1
4.9
L
SC
SCL
L
SC
L
SL
SL
L
SL
SL
SL
SL
SL
SL
Sl
SL
SL
SL
SL
SL
SL
SL
SL
SL
14.0
15.6
15.9
13.8
64.5
17.2
11.4
17.7
29.4
51.1
11.3
15.2
14.9
13.0
11.3
11.9
11.9
13.1
10.8
12.1
12.7
9.8
11.7
11.4
12.8
16.5
15.8
SL
CL
6.3
9.4
6.5
5.6
7.2
28.5
id
id
N
(g kgÿ1)
C/N
(ratio)
pH
(H2O)
CaCO3
(g kgÿ1)
CEC
(cmol+ kgÿ1)
1.43
1.70
1.83
1.30
6.52
1.37
1.24
1.93
2.59
3.44
1.20
1.45
1.42
1.24
1.23
1.31
1.25
1.35
1.16
1.21
1.27
1.03
1.23
1.23
1.39
9.8
9.2
8.7
10.6
9.9
12.6
9.2
9.2
11.4
14.9
9.4
10.5
10.5
10.5
9.2
9.1
9.5
9.7
9.3
10.0
10.0
9.5
9.5
9.3
9.2
7.9
7.2
8.2
7.0
7.8
7.1
6.2
5.6
5.7
5.5
8.2
8.0
8.0
8.1
8.0
7.9
7.2
7.5
6.4
6.4
6.6
6.1
6.3
6.8
6.5
11
0
195
0
19
0
0
0
0
0
35
19
18
26
6
6
1
4
0
0
0
0
0
0
0
11.0
23.4
14.5
9.1
38.0
9.1
7.3
9.7
13.0
15.3
15.6
16.9
17.2
16.5
12.7
13.1
12.3
12.1
11.8
12.2
12.0
10.5
11.2
12.8
13.4
18.3
39.1
1.70
4.32
10.8
9.03
8.0
8.0
33
174
17.7
24.3
CL
CL
CL
SC
SC
20.2
25.4
22.7
32.1
28.2
2.36
3.02
2.68
3.85
3.19
8.56
8.41
8.47
8.34
8.84
8.1
8.0
8.0
8.0
7.9
83
23
52
99
32
20.0
21.5
20.4
25.4
19.6
C
id
id
21.7
22.8
20.2
ndb
nd
nd
6.2
6.6
6.1
0
0
0
17.8
18.1
16.5
sand
nd
nd
nd
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
French soils
Plots frequently receiving atrazine (continuous maize or rotation including maize)
Citeaux
R
M
18.0
50.0
Baccon
R
M
37.1
57.1
Montoison
C
M
20.6
17.9
CoÃte Saint AndreÂ
C
M
18.2
38.7
Chigy
C
M
41.2
51.0
Chabeuil
C
M
15.8
36.1
Le Rheu
R
M
16.6
70.7
Loustalet
C
M
19.2
65.7
Plumelec
R
M
16.1
48.2
Salinis
C
M
25.3
57.0
Grignon 1: DeheÂrain
C
M
20.3
61.3
Grignon 2: Plateau
R
M
22.5
60.2
Grignon 3: Plateau
R
W
22.3
61.1
Grignon 4: Plateau
C
M
22.1
64.6
Grignon 5: Block 1 untreated
C
M
20.1
75.0
Grignon 6: Block 1 sludge
C
M
16.8
77.8
Grignon 7: Block 2 untreated
C
M
17.2
77.9
Grignon 8: Block 2 untreated
C
M
17.1
78.4
Grignon 9: Block 2 untreated
C
M
17.5
78.1
Grignon 10: Block 2 mineral N
C
M
15.9
79.8
Grignon 11: Block 2 sludge
C
M
17.0
78.7
Grignon 12: Block 3 untreated
C
M
15.1
79.6
Grignon 13: Block 3 sludge
C
M
15.9
78.9
Grignon 14: Block 4 untreated
C
M
18.3
76.5
Grignon 15: Block 4 sludge
C
M
19.1
75.9
Plots never receiving atrazine
Grignon 16: plateau
C
W
21.0
59.1
Boyer
C
G
39.5
26.4
Plots under continuous maize since various length of time and on the same type of soil
Boyer: maize for 21 yr
C
M
33.6
51.5
Boyer: maize for 8 yr
C
M
36.6
51.6
Boyer: maize for 5 yr
C
M
35.3
52.8
Boyer: maize for 3 yr
C
M
44.2
39.8
Boyer: maize for 2 yr
C
M
32.7
56.8
Texture
a
b
Management: C=continuous; R=rotation. Crop: M=maize; W=wheat; G=grass. Texture: L=loam; SC=silty clay; SCL=sandy clay loam; SL=silt loam; CL=clay loam.
nd: not determined.
c
id: identical to the previous one. Only one particle size analysis was done in Harrow and Winchester, assuming that the dierent plots had the same particle size distribution.
21.5
20.9
0
0
6.1
6.3
nd
nd
nd
nd
22.9
21.8
272
id
G
G
344
id
384
id
CL
id
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
id
id
id
id
id
id
27.2
id
id
id
Harrow 4
C
Harrow 5
C
Harrow 6
C
Harrow 7
C
Harrow 8
R
Harrow 9
R
Winchester 1
C
Winchester 2
C
Winchester 3
C
Winchester 4
C
Plots with last atrazine application in 1990
Winchester 5
C
Winchester 6
C
M
M
M
M
M
M
M
M
M
M
id
id
id
id
id
id
34.4
id
id
id
id
id
id
id
id
id
38.4
id
id
id
id
id
id
id
id
id
CL
id
id
id
16.5
18.4
18.2
21.9
19.8
20.0
22.5
37.8
28.8
32.7
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
7.0
5.9
6.4
6.3
6.5
6.3
6.5
6.3
6.4
6.4
0
0
0
0
0
0
0
0
0
0
18.0
17.7
18.6
18.1
18.2
17.8
16.1
26.4
20.7
23.5
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
617
hydrolyzed to biuret and then to urea (Cook, 1987).
Atrazine is converted by ring-mineralizing bacteria to
cyanuric acid through a series of hydrolytic reactions
starting with dechlorination and then the sequential
removal of the two alkylamino chains (Mandelbaum et
al., 1995; de Souza et al., 1995; Boundy-Mills et al.,
1997).
We previously demonstrated that the rate of atrazine
mineralization increased with the frequency of ®eld application of the herbicide (Barriuso and Houot, 1996),
a result recently con®rmed by Vanderheyden et al.
(1997) and Pussemier et al. (1997) who similarly found
rapid dissipation of atrazine in numerous soils frequently treated with atrazine under ®eld conditions.
The present work was undertaken to broaden the
type of soils tested for accelerated degradation of atrazine, determine if there was a correlation between the
development of accelerated degradation and selected
soil physical or chemical properties and to determine
the minimum number of ®eld applications of the herbicide required to stimulate accelerated degradation.
2. Materials and methods
2.1. Soils and soil management
Thirty-two soils were sampled from 12 locations in
France and 15 soils were sampled from two locations
in Ontario, Canada (Table 1). The soils were chosen to
encompass a wide range of physical and chemical
properties and atrazine-treatment histories. Sixteen of
the French soil samples came from three ®eld experiments located near Grignon. One soil was sampled
from the long-term ``DeheÂrain'' experiment, comparing
the eect of dierent fertilization regimes on maize
yield (described in Houot and Chaussod, 1995). Four
soils were sampled from the ``Plateau'' experiment,
comparing yield in crop rotations consisting of maizewheat rotation, continuous maize or continuous
wheat. Atrazine behaviour has previously been studied
in these soils (Barriuso and Houot, 1996). Eleven soils
were sampled in a third, as yet unpublished, ®eld experiment studying the eect of sewage sludge application on maize production. The treatments are
arranged in a series of random plots on four adjacent
®eld blocks. Six untreated plots, four sludge-treated
plots and one plot receiving mineral N fertilizer were
sampled. Finally, a series of plots of uniform soil type
near Boyer were sampled. Plots, initially under continuous grass pasture, are progressively being converted to continuous maize, providing a range of wellcharacterized atrazine treatment histories. Six plots
were sampled, one still in pasture, the others having
been continuously cropped to maize for 2, 3, 5, 8 or
21 yr. Atrazine was applied annually on all plots
618
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
cropped to maize at the rate of 1 kg haÿ1. Therefore
all of the French soils except the continuous wheat
plot in Grignon and the permanent grass plot in Boyer
have received atrazine frequently under ®eld conditions.
The Canadian soils were obtained near the towns of
Winchester and Harrow. All the soils had been treated
with atrazine annually or biannually during the previous 5 yr, except for two of the Winchester soils for
which the last atrazine application occurred 6 yr prior
to the sampling. Details on soil management are provided in Table 1.
The soils were all sampled during the autumn of
1996 or spring of 1997, before atrazine application, in
the case of the sludge application experiment. Samples
(0±20 cm depth), were sieved (5 mm) and kept at 48C
for a maximum of 14 d before starting the incubations.
Soil water content was measured gravimetrically at
1058C.
Physico-chemical analysis of the soils are reported in
Table 1. Particle size fractionation and analysis was
realized using the pipette method after destruction of
organic matter and carbonates (Day, 1965). Total organic carbon was determined by sulfochromic oxidation and total nitrogen using the classical Kjeldahl
method as described in the norm ISO 11261 (AFNOR,
1996). The volumetric method as described in the
norm ISO 10693 (AFNOR, 1996) was used to measure
the carbonate content of the soils. Soil pH was
measured in a water suspension (1/5, v/v) as described
in the norm ISO 10390 (AFNOR, 1996). The soil cation exchange capacity was measured with ammonium
acetate as described by Chapman (1965).
2.2. Chemicals
Analytical standards of atrazine and its metabolites
were purchased from ChemService (West Chester, PA,
USA). The [U-ring-14C] atrazine was purchased from
Amersham (Buckinghamshire, UK, speci®c activity
659 MBq mmolÿ1; radiochemical purity of 97%). Two
solutions of 14C-atrazine were prepared: solution A in
water at 10 mg lÿ1 and 8.8 MBq lÿ1 and solution B in
10 mM CaCl2 at 9.85 mg lÿ1 and 0.17 MBq mlÿ1.
2.3. Atrazine mineralization incubations
Fresh moist soil equivalent to 20 g dry weight was
dispensed into triplicate 500 ml jars. The soil water
content was adjusted to the humidity equivalent to the
matric potential of ÿ104 Pa, using 1 ml of the solution
A of atrazine, supplemented with additional deionized
water when necessary. The ®nal atrazine concentration
was 0.5 mg of atrazine kgÿ1 soil, equivalent to 0.44
MBq kgÿ1 soil. The jars each received a CO2 trap consisting of a vial containing 5 ml of 0.5 M NaOH and
were hermetically sealed and incubated statically at 28
2 18C for 35 d. The NaOH was periodically sampled
and replaced. The mineralized 14C-CO2 was measured
in 0.5 ml of the traps added of 4 ml of scintillation
cocktail (Ultima Gold XR, Packard), after luminescence extinction, with a scintillation counter Betamatic
V (Kontron Ins., Montigny le Bretonneux, France).
2.4. Analysis of the distribution of
14
C-atrazine residues
At the end of the 35 d incubation, the distribution
of the remaining extractable and non-extractable
radioactivity was determined as follows in the French
soils (the soils sampled in the sludge experiment were
not extracted). Each sample was extracted in 100 ml of
an aqueous solution of 10 mM CaCl2 for 24 h. The
supernatant was recovered by centrifugation at 8000 g
for 15 min. The solids were further extracted for 24 h
with 100 ml of methanol, three successive times. The
extractable radioactivity in the aqueous and the methanol extracts was determined by liquid scintillation
counting. The non-extractable radioactivity, corresponding to the ``bound residue'' fraction, was
measured by scintillation counting of the 14CO2
evolved after combustion of the solids recovered after
methanol extraction (Sample Oxidizer 307, Packard,
Meriden, CT, USA). The water extracts were concentrated with LiChrolut EN-(200 mg) cartridges (Merck,
Darmstadt, Germany) eluted with 5 ml of methanol,
then evaporated until dryness under vacuum with a
Rotavapor RE 111 (BuÈchi, Flawil, Switzerland). The
residue was then dissolved in 1 ml of the solvent used
for the HPLC analysis. The methanol extracts were
concentrated until dryness by evaporation under vacuum and the residue dissolved in 3 ml of the HPLC
solvent. Samples for HPLC analyses were ®ltered
(Cameo 13N nylon membrane, 0.45 mm pore size,
MSI, Westboro, MA, USA). 14C-atrazine and 14Cmetabolites were analyzed using a Waters HPLC
instrument (600E Multisolvent Delivery System, 717
Autosampler and a Novapak C18 column of 5 mm
and 250 4.6 mm; Waters, Milford, MA, USA)
equipped with a diode array detector (Waters 916)
coupled online with a radioactive ¯ow detector (Packard-Radiomatic Flo-one A550). The mobile phase was
methanol±water buered with 50 mM ammonium
acetate with pH adjusted to 7.4. The gradient chromatography started with 40/60 methanol±water (vol/vol),
reaching 80/20 methanol±water after 20 min with a
concave gradient (gradient 7 in Waters software),
returning to 40/60 methanol±water with a linear gradient (gradient 6 in Waters software) after 21 min and
remained constant until 35 min. The mobile phase ¯ow
was 1.0 ml minÿ1 and the injection volume was 800 ml.
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
2.5. Microbial activity and biomass
Total microbial biomass was estimated in fresh soil
samples with the fumigation-extraction method (Vance
et al., 1987). The amounts of carbon and nitrogen
mineralized during the atrazine incubations was determined as a measure of microbial activity. Total C-CO2
evolved during the incubations was measured in the
NaOH traps by colorimetry on a continuous ¯ow analyzer (Skalar, the Netherlands). The nitrogen mineralized during the incubations was calculated from the
dierence of concentration in mineral N in CaCl2
extracts taken at the beginning and at the end of the
incubations. Mineral N in the extracts was measured
by colorimetry on the continuous ¯ow analyzer.
2.6. Determination of sorption coecients
All measurements were made in duplicate. Three
grams of air dried soil were mixed with 15 ml of solution B of atrazine in 25 ml Corex centrifuge tubes
with Te¯on caps. The suspensions were agitated for 24
h at 20 2 18C. Then the mixtures were centrifuged at
10,000 g for 15 min. The radioactivity was measured
in 0.5 ml of the supernatant added of 4 ml of scintillation liquid with the scintillation counter. The amount
of adsorbed atrazine was calculated from the dierence
between the initial herbicide concentration and the
equilibrium concentration. The Kd sorption coecients
were calculated as the ratio of the amount of adsorbed
herbicide (mg) per unit mass of soil (kg) to the equilibrium concentration (mg lÿ1). The sorption coecients
relative to the soil organic carbon (Koc) were obtained
by dividing the Kd values by the organic carbon content of the soils.
2.7. Statistical analysis
The STAT-ITCF software was used for statistical
analysis. A correlation matrix was obtained using the
following parameters: the proportion of added atrazine
mineralized at the end of the incubations, the soil
characteristics presented in Table 1, the sorption coecients and the variables describing global microbial activity. Only data obtained from the French and
Canadian soils which had been frequently treated with
atrazine were used for the statistical analysis for two
reasons: (1) repeated ®eld applications of atrazine had
been shown to induce accelerated atrazine mineralization in relation with the soil micro¯ora acclimatization; (2) we wanted to determine if accelerated
mineralization of atrazine was related to soil physicochemical characteristics. The French and Canadian
soils classi®ed as frequently receiving atrazine in
Table 1 were used, in addition to the French soil
``Boyer: maize for 21 yr'' (39 soils all together).
619
3. Results and discussion
3.1. Soil diversity
The collection of soils obtained varied widely with
respect to their physical and chemical properties
(Table 1). Among the French soils, the range of physico-chemical characteristics was rather large, with
dierent textures, organic matter contents and pH
values. The 16 soils sampled in Grignon all had similar
texture and mainly varied from their carbonate content
and pH.
The ®ve soils sampled in Boyer varied mainly in
their organic C and N contents which decreased with
the duration of maize culture as classically observed
during the ®rst years of cultivation of soils previously
under permanent grass (Arrouays and Pelissier, 1994).
Of the Canadian soils, the Harrow soils were clays;
clay loams were obtained from the Winchester area.
Dierent crop management resulted in dierences in
the organic carbon contents. Some dierences were
also observed in soil pH.
3.2. Total microbial activity
Microbial biomass measurements and total C and N
mineralization during the incubations were used as indicators of the total microbial activity in the soils
(Table 2). In most soils, the size of the microbial biomass corresponded to about 2% of the total organic
carbon, a proportion frequently encountered (Jenkinson and Ladd, 1981). This proportion decreased to
below 1% in the Salinis and Plumelec soils, probably
because of the acidic pH (Anderson and Domsch,
1993). The size of the microbial biomass increased signi®cantly with clay content r 0:691, P < 0.01;
Table 3), in agreement with the results of Chaussod et
al. (1986). There was also a signi®cant but weaker relationship of microbial biomass with total soil organic
carbon of the soils r 0:403, P < 0.05; Table 3). Carbon mineralization was not correlated with the soil organic carbon r 0:131; Table 3) but N mineralization
was r 0:454, P < 0.01). Both C and N mineralization increased signi®cantly with soil clay content (r =
0.730 and 0.413, respectively for C and N mineralization). On the other hand, soil pH was correlated positively with C mineralization r 0:661, P < 0.01) but
negatively
correlated
with
N
mineralization
r ÿ0:483, P
www.elsevier.com/locate/soilbio
Dependence of accelerated degradation of atrazine on soil pH in
French and Canadian soils
Sabine Houot a,*, Edward Topp b, Abdellah Yassir c, 1, Guy Soulas c
a
I.N.R.A. Unite de Science du Sol, BP 01, 78850 Thiverval-Grignon, France
Pest Management Research Centre, Agriculture and Agrifood Canada, 1391 Sandford Street, London, Ontario, Canada N5V 4T3
c
I.N.R.A.-C.M.S.E., Laboratoire de Microbiologie des Sols, 17 rue Sully, BV 1540, 21034 Dijon Cedex, France
b
Accepted 30 September 1999
Abstract
A series of agricultural soils varying in their atrazine treatment history were sampled from 12 sites in France and two sites in
Canada. The soils varied widely with respect to soil chemical, physical and microbiological (total microbial biomass, kinetics of
C and N mineralization) properties. Soils treated with as few as two successive atrazine ®eld applications mineralized [Uring-14C]atrazine signi®cantly more rapidly in 35 d laboratory incubations than did soils which had never received atrazine.
Longer treatment history tended to favour more rapid mineralization in the so-called ``adapted'' soils. Up to 80% of the initially
applied 14C-atrazine was mineralized at the end of the incubations in these adapted soils. Of the properties tested, soil pH was
the most signi®cantly related to atrazine mineralized. In soils with pH lower than 6.5, less than 25% of the initial 14C-atrazine
was mineralized even after repeated application in ®eld conditions. Atrazine retention in soil did not in¯uence its mineralization
rate. Both hydroxylated and dealkylated atrazine metabolites were detected, but no clear pattern of metabolite production could
be determined. Large amounts of bound residues were formed in soils that mineralized little atrazine. 7 2000 Elsevier Science
Ltd. All rights reserved.
Keywords: Atrazine; Accelerated mineralization; Soil; pH
1. Introduction
Repeated application of some xenobiotic pesticides
to soil can result in the adaptation and development of
a soil micro¯ora which can rapidly metabolize the
compound (Racke and Coats, 1990). In some cases
accelerated degradation can result in loss of ecacy
and signi®cant economic cost (Suett et al., 1996a).
Accelerated degradation of a number of herbicides,
including phenoxyalkanoic acids, thiocarbamates
(Roeth, 1986) linuron, propyzamide, metamitrone and
* Corresponding author. Tel.: +33-1-3081-5401; fax: +33-1-30815396.
E-mail address: [email protected] (S. Houot).
1
Present address: Universite Caddi-Ayyad, Faculte des Sciences
Semlalia, DeÂpartement de Biologie, BPS 15, Marrakech, Maroc.
napropamide (Walker and Welch, 1992) has been
documented. Atrazine [6-chloro-N2-ethyl-N4-isopropyl-1.3.5-triazine-2.4-diamine] is a herbicide widely
used in maize production. It was considered relatively
recalcitrant in soils, although microbial degradation
has always been recognized as the principal mechanism
of atrazine dissipation in soils (Kaufman and Kearney,
1970). Until recently, N-dealkylation reactions catalyzed by bacteria or fungi was the classical pathway of
microbial degradation described (Kaufman and Kearney, 1970; Mougin et al., 1994; Nagy et al., 1995).
Complete and rapid mineralization of the 14C-labelled
s-triazine ring, both by soil populations and by bacterial isolates, is now commonly observed (Assaf and
Turco, 1994; Yanze-Kontchou and Gschwind, 1994;
Mandelbaum et al., 1995; Radosevitch et al., 1995;
Barriuso and Houot, 1996; Vanderheyden et al., 1997).
The ring-cleavage substrate is cyanuric acid, which is
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 8 8 - 1
616
Table 1
Location, cropping and management, and primary physical and chemical properties (expressed on a dry weight basis) of the soils used in this studya
Location
Management and crop in 1996
Particle size (%)
clay
silt
Canadian soils
Plots frequently receiving atrazine (continuous maize or rotation including maize)
Harrow 1
R
M
47.3
Harrow 2
R
M
idc
Harrow 3
C
M
id
24.2
id
id
C
(g kgÿ1)
30.9
5.7
41.4
43.0
5.8
48.0
12.6
15.0
35.6
17.6
14.8
15.3
14.7
10.6
4.3
4.8
4.5
4.1
4.3
4.1
4.1
5.2
5.1
5.1
4.9
L
SC
SCL
L
SC
L
SL
SL
L
SL
SL
SL
SL
SL
SL
Sl
SL
SL
SL
SL
SL
SL
SL
SL
SL
14.0
15.6
15.9
13.8
64.5
17.2
11.4
17.7
29.4
51.1
11.3
15.2
14.9
13.0
11.3
11.9
11.9
13.1
10.8
12.1
12.7
9.8
11.7
11.4
12.8
16.5
15.8
SL
CL
6.3
9.4
6.5
5.6
7.2
28.5
id
id
N
(g kgÿ1)
C/N
(ratio)
pH
(H2O)
CaCO3
(g kgÿ1)
CEC
(cmol+ kgÿ1)
1.43
1.70
1.83
1.30
6.52
1.37
1.24
1.93
2.59
3.44
1.20
1.45
1.42
1.24
1.23
1.31
1.25
1.35
1.16
1.21
1.27
1.03
1.23
1.23
1.39
9.8
9.2
8.7
10.6
9.9
12.6
9.2
9.2
11.4
14.9
9.4
10.5
10.5
10.5
9.2
9.1
9.5
9.7
9.3
10.0
10.0
9.5
9.5
9.3
9.2
7.9
7.2
8.2
7.0
7.8
7.1
6.2
5.6
5.7
5.5
8.2
8.0
8.0
8.1
8.0
7.9
7.2
7.5
6.4
6.4
6.6
6.1
6.3
6.8
6.5
11
0
195
0
19
0
0
0
0
0
35
19
18
26
6
6
1
4
0
0
0
0
0
0
0
11.0
23.4
14.5
9.1
38.0
9.1
7.3
9.7
13.0
15.3
15.6
16.9
17.2
16.5
12.7
13.1
12.3
12.1
11.8
12.2
12.0
10.5
11.2
12.8
13.4
18.3
39.1
1.70
4.32
10.8
9.03
8.0
8.0
33
174
17.7
24.3
CL
CL
CL
SC
SC
20.2
25.4
22.7
32.1
28.2
2.36
3.02
2.68
3.85
3.19
8.56
8.41
8.47
8.34
8.84
8.1
8.0
8.0
8.0
7.9
83
23
52
99
32
20.0
21.5
20.4
25.4
19.6
C
id
id
21.7
22.8
20.2
ndb
nd
nd
6.2
6.6
6.1
0
0
0
17.8
18.1
16.5
sand
nd
nd
nd
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
French soils
Plots frequently receiving atrazine (continuous maize or rotation including maize)
Citeaux
R
M
18.0
50.0
Baccon
R
M
37.1
57.1
Montoison
C
M
20.6
17.9
CoÃte Saint AndreÂ
C
M
18.2
38.7
Chigy
C
M
41.2
51.0
Chabeuil
C
M
15.8
36.1
Le Rheu
R
M
16.6
70.7
Loustalet
C
M
19.2
65.7
Plumelec
R
M
16.1
48.2
Salinis
C
M
25.3
57.0
Grignon 1: DeheÂrain
C
M
20.3
61.3
Grignon 2: Plateau
R
M
22.5
60.2
Grignon 3: Plateau
R
W
22.3
61.1
Grignon 4: Plateau
C
M
22.1
64.6
Grignon 5: Block 1 untreated
C
M
20.1
75.0
Grignon 6: Block 1 sludge
C
M
16.8
77.8
Grignon 7: Block 2 untreated
C
M
17.2
77.9
Grignon 8: Block 2 untreated
C
M
17.1
78.4
Grignon 9: Block 2 untreated
C
M
17.5
78.1
Grignon 10: Block 2 mineral N
C
M
15.9
79.8
Grignon 11: Block 2 sludge
C
M
17.0
78.7
Grignon 12: Block 3 untreated
C
M
15.1
79.6
Grignon 13: Block 3 sludge
C
M
15.9
78.9
Grignon 14: Block 4 untreated
C
M
18.3
76.5
Grignon 15: Block 4 sludge
C
M
19.1
75.9
Plots never receiving atrazine
Grignon 16: plateau
C
W
21.0
59.1
Boyer
C
G
39.5
26.4
Plots under continuous maize since various length of time and on the same type of soil
Boyer: maize for 21 yr
C
M
33.6
51.5
Boyer: maize for 8 yr
C
M
36.6
51.6
Boyer: maize for 5 yr
C
M
35.3
52.8
Boyer: maize for 3 yr
C
M
44.2
39.8
Boyer: maize for 2 yr
C
M
32.7
56.8
Texture
a
b
Management: C=continuous; R=rotation. Crop: M=maize; W=wheat; G=grass. Texture: L=loam; SC=silty clay; SCL=sandy clay loam; SL=silt loam; CL=clay loam.
nd: not determined.
c
id: identical to the previous one. Only one particle size analysis was done in Harrow and Winchester, assuming that the dierent plots had the same particle size distribution.
21.5
20.9
0
0
6.1
6.3
nd
nd
nd
nd
22.9
21.8
272
id
G
G
344
id
384
id
CL
id
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
id
id
id
id
id
id
27.2
id
id
id
Harrow 4
C
Harrow 5
C
Harrow 6
C
Harrow 7
C
Harrow 8
R
Harrow 9
R
Winchester 1
C
Winchester 2
C
Winchester 3
C
Winchester 4
C
Plots with last atrazine application in 1990
Winchester 5
C
Winchester 6
C
M
M
M
M
M
M
M
M
M
M
id
id
id
id
id
id
34.4
id
id
id
id
id
id
id
id
id
38.4
id
id
id
id
id
id
id
id
id
CL
id
id
id
16.5
18.4
18.2
21.9
19.8
20.0
22.5
37.8
28.8
32.7
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
7.0
5.9
6.4
6.3
6.5
6.3
6.5
6.3
6.4
6.4
0
0
0
0
0
0
0
0
0
0
18.0
17.7
18.6
18.1
18.2
17.8
16.1
26.4
20.7
23.5
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
617
hydrolyzed to biuret and then to urea (Cook, 1987).
Atrazine is converted by ring-mineralizing bacteria to
cyanuric acid through a series of hydrolytic reactions
starting with dechlorination and then the sequential
removal of the two alkylamino chains (Mandelbaum et
al., 1995; de Souza et al., 1995; Boundy-Mills et al.,
1997).
We previously demonstrated that the rate of atrazine
mineralization increased with the frequency of ®eld application of the herbicide (Barriuso and Houot, 1996),
a result recently con®rmed by Vanderheyden et al.
(1997) and Pussemier et al. (1997) who similarly found
rapid dissipation of atrazine in numerous soils frequently treated with atrazine under ®eld conditions.
The present work was undertaken to broaden the
type of soils tested for accelerated degradation of atrazine, determine if there was a correlation between the
development of accelerated degradation and selected
soil physical or chemical properties and to determine
the minimum number of ®eld applications of the herbicide required to stimulate accelerated degradation.
2. Materials and methods
2.1. Soils and soil management
Thirty-two soils were sampled from 12 locations in
France and 15 soils were sampled from two locations
in Ontario, Canada (Table 1). The soils were chosen to
encompass a wide range of physical and chemical
properties and atrazine-treatment histories. Sixteen of
the French soil samples came from three ®eld experiments located near Grignon. One soil was sampled
from the long-term ``DeheÂrain'' experiment, comparing
the eect of dierent fertilization regimes on maize
yield (described in Houot and Chaussod, 1995). Four
soils were sampled from the ``Plateau'' experiment,
comparing yield in crop rotations consisting of maizewheat rotation, continuous maize or continuous
wheat. Atrazine behaviour has previously been studied
in these soils (Barriuso and Houot, 1996). Eleven soils
were sampled in a third, as yet unpublished, ®eld experiment studying the eect of sewage sludge application on maize production. The treatments are
arranged in a series of random plots on four adjacent
®eld blocks. Six untreated plots, four sludge-treated
plots and one plot receiving mineral N fertilizer were
sampled. Finally, a series of plots of uniform soil type
near Boyer were sampled. Plots, initially under continuous grass pasture, are progressively being converted to continuous maize, providing a range of wellcharacterized atrazine treatment histories. Six plots
were sampled, one still in pasture, the others having
been continuously cropped to maize for 2, 3, 5, 8 or
21 yr. Atrazine was applied annually on all plots
618
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
cropped to maize at the rate of 1 kg haÿ1. Therefore
all of the French soils except the continuous wheat
plot in Grignon and the permanent grass plot in Boyer
have received atrazine frequently under ®eld conditions.
The Canadian soils were obtained near the towns of
Winchester and Harrow. All the soils had been treated
with atrazine annually or biannually during the previous 5 yr, except for two of the Winchester soils for
which the last atrazine application occurred 6 yr prior
to the sampling. Details on soil management are provided in Table 1.
The soils were all sampled during the autumn of
1996 or spring of 1997, before atrazine application, in
the case of the sludge application experiment. Samples
(0±20 cm depth), were sieved (5 mm) and kept at 48C
for a maximum of 14 d before starting the incubations.
Soil water content was measured gravimetrically at
1058C.
Physico-chemical analysis of the soils are reported in
Table 1. Particle size fractionation and analysis was
realized using the pipette method after destruction of
organic matter and carbonates (Day, 1965). Total organic carbon was determined by sulfochromic oxidation and total nitrogen using the classical Kjeldahl
method as described in the norm ISO 11261 (AFNOR,
1996). The volumetric method as described in the
norm ISO 10693 (AFNOR, 1996) was used to measure
the carbonate content of the soils. Soil pH was
measured in a water suspension (1/5, v/v) as described
in the norm ISO 10390 (AFNOR, 1996). The soil cation exchange capacity was measured with ammonium
acetate as described by Chapman (1965).
2.2. Chemicals
Analytical standards of atrazine and its metabolites
were purchased from ChemService (West Chester, PA,
USA). The [U-ring-14C] atrazine was purchased from
Amersham (Buckinghamshire, UK, speci®c activity
659 MBq mmolÿ1; radiochemical purity of 97%). Two
solutions of 14C-atrazine were prepared: solution A in
water at 10 mg lÿ1 and 8.8 MBq lÿ1 and solution B in
10 mM CaCl2 at 9.85 mg lÿ1 and 0.17 MBq mlÿ1.
2.3. Atrazine mineralization incubations
Fresh moist soil equivalent to 20 g dry weight was
dispensed into triplicate 500 ml jars. The soil water
content was adjusted to the humidity equivalent to the
matric potential of ÿ104 Pa, using 1 ml of the solution
A of atrazine, supplemented with additional deionized
water when necessary. The ®nal atrazine concentration
was 0.5 mg of atrazine kgÿ1 soil, equivalent to 0.44
MBq kgÿ1 soil. The jars each received a CO2 trap consisting of a vial containing 5 ml of 0.5 M NaOH and
were hermetically sealed and incubated statically at 28
2 18C for 35 d. The NaOH was periodically sampled
and replaced. The mineralized 14C-CO2 was measured
in 0.5 ml of the traps added of 4 ml of scintillation
cocktail (Ultima Gold XR, Packard), after luminescence extinction, with a scintillation counter Betamatic
V (Kontron Ins., Montigny le Bretonneux, France).
2.4. Analysis of the distribution of
14
C-atrazine residues
At the end of the 35 d incubation, the distribution
of the remaining extractable and non-extractable
radioactivity was determined as follows in the French
soils (the soils sampled in the sludge experiment were
not extracted). Each sample was extracted in 100 ml of
an aqueous solution of 10 mM CaCl2 for 24 h. The
supernatant was recovered by centrifugation at 8000 g
for 15 min. The solids were further extracted for 24 h
with 100 ml of methanol, three successive times. The
extractable radioactivity in the aqueous and the methanol extracts was determined by liquid scintillation
counting. The non-extractable radioactivity, corresponding to the ``bound residue'' fraction, was
measured by scintillation counting of the 14CO2
evolved after combustion of the solids recovered after
methanol extraction (Sample Oxidizer 307, Packard,
Meriden, CT, USA). The water extracts were concentrated with LiChrolut EN-(200 mg) cartridges (Merck,
Darmstadt, Germany) eluted with 5 ml of methanol,
then evaporated until dryness under vacuum with a
Rotavapor RE 111 (BuÈchi, Flawil, Switzerland). The
residue was then dissolved in 1 ml of the solvent used
for the HPLC analysis. The methanol extracts were
concentrated until dryness by evaporation under vacuum and the residue dissolved in 3 ml of the HPLC
solvent. Samples for HPLC analyses were ®ltered
(Cameo 13N nylon membrane, 0.45 mm pore size,
MSI, Westboro, MA, USA). 14C-atrazine and 14Cmetabolites were analyzed using a Waters HPLC
instrument (600E Multisolvent Delivery System, 717
Autosampler and a Novapak C18 column of 5 mm
and 250 4.6 mm; Waters, Milford, MA, USA)
equipped with a diode array detector (Waters 916)
coupled online with a radioactive ¯ow detector (Packard-Radiomatic Flo-one A550). The mobile phase was
methanol±water buered with 50 mM ammonium
acetate with pH adjusted to 7.4. The gradient chromatography started with 40/60 methanol±water (vol/vol),
reaching 80/20 methanol±water after 20 min with a
concave gradient (gradient 7 in Waters software),
returning to 40/60 methanol±water with a linear gradient (gradient 6 in Waters software) after 21 min and
remained constant until 35 min. The mobile phase ¯ow
was 1.0 ml minÿ1 and the injection volume was 800 ml.
S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625
2.5. Microbial activity and biomass
Total microbial biomass was estimated in fresh soil
samples with the fumigation-extraction method (Vance
et al., 1987). The amounts of carbon and nitrogen
mineralized during the atrazine incubations was determined as a measure of microbial activity. Total C-CO2
evolved during the incubations was measured in the
NaOH traps by colorimetry on a continuous ¯ow analyzer (Skalar, the Netherlands). The nitrogen mineralized during the incubations was calculated from the
dierence of concentration in mineral N in CaCl2
extracts taken at the beginning and at the end of the
incubations. Mineral N in the extracts was measured
by colorimetry on the continuous ¯ow analyzer.
2.6. Determination of sorption coecients
All measurements were made in duplicate. Three
grams of air dried soil were mixed with 15 ml of solution B of atrazine in 25 ml Corex centrifuge tubes
with Te¯on caps. The suspensions were agitated for 24
h at 20 2 18C. Then the mixtures were centrifuged at
10,000 g for 15 min. The radioactivity was measured
in 0.5 ml of the supernatant added of 4 ml of scintillation liquid with the scintillation counter. The amount
of adsorbed atrazine was calculated from the dierence
between the initial herbicide concentration and the
equilibrium concentration. The Kd sorption coecients
were calculated as the ratio of the amount of adsorbed
herbicide (mg) per unit mass of soil (kg) to the equilibrium concentration (mg lÿ1). The sorption coecients
relative to the soil organic carbon (Koc) were obtained
by dividing the Kd values by the organic carbon content of the soils.
2.7. Statistical analysis
The STAT-ITCF software was used for statistical
analysis. A correlation matrix was obtained using the
following parameters: the proportion of added atrazine
mineralized at the end of the incubations, the soil
characteristics presented in Table 1, the sorption coecients and the variables describing global microbial activity. Only data obtained from the French and
Canadian soils which had been frequently treated with
atrazine were used for the statistical analysis for two
reasons: (1) repeated ®eld applications of atrazine had
been shown to induce accelerated atrazine mineralization in relation with the soil micro¯ora acclimatization; (2) we wanted to determine if accelerated
mineralization of atrazine was related to soil physicochemical characteristics. The French and Canadian
soils classi®ed as frequently receiving atrazine in
Table 1 were used, in addition to the French soil
``Boyer: maize for 21 yr'' (39 soils all together).
619
3. Results and discussion
3.1. Soil diversity
The collection of soils obtained varied widely with
respect to their physical and chemical properties
(Table 1). Among the French soils, the range of physico-chemical characteristics was rather large, with
dierent textures, organic matter contents and pH
values. The 16 soils sampled in Grignon all had similar
texture and mainly varied from their carbonate content
and pH.
The ®ve soils sampled in Boyer varied mainly in
their organic C and N contents which decreased with
the duration of maize culture as classically observed
during the ®rst years of cultivation of soils previously
under permanent grass (Arrouays and Pelissier, 1994).
Of the Canadian soils, the Harrow soils were clays;
clay loams were obtained from the Winchester area.
Dierent crop management resulted in dierences in
the organic carbon contents. Some dierences were
also observed in soil pH.
3.2. Total microbial activity
Microbial biomass measurements and total C and N
mineralization during the incubations were used as indicators of the total microbial activity in the soils
(Table 2). In most soils, the size of the microbial biomass corresponded to about 2% of the total organic
carbon, a proportion frequently encountered (Jenkinson and Ladd, 1981). This proportion decreased to
below 1% in the Salinis and Plumelec soils, probably
because of the acidic pH (Anderson and Domsch,
1993). The size of the microbial biomass increased signi®cantly with clay content r 0:691, P < 0.01;
Table 3), in agreement with the results of Chaussod et
al. (1986). There was also a signi®cant but weaker relationship of microbial biomass with total soil organic
carbon of the soils r 0:403, P < 0.05; Table 3). Carbon mineralization was not correlated with the soil organic carbon r 0:131; Table 3) but N mineralization
was r 0:454, P < 0.01). Both C and N mineralization increased signi®cantly with soil clay content (r =
0.730 and 0.413, respectively for C and N mineralization). On the other hand, soil pH was correlated positively with C mineralization r 0:661, P < 0.01) but
negatively
correlated
with
N
mineralization
r ÿ0:483, P