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Soil Biology & Biochemistry 32 (2000) 389±401
www.elsevier.com/locate/soilbio

Dependence of atrazine degradation on C and N availability in
adapted and non-adapted soils
Rahima Abdelha®d, Sabine Houot*, Enrique Barriuso
I.N.R.A., Unite de Science du Sol, BP 01, 78850 Thiverval-Grignon, France
Accepted 23 August 1999

Abstract
Atrazine degradation in soil was a€ected by microbial adaptation and C and N availability. Accelerated atrazine
mineralization was observed after repeated applications to a soil under continuous maize (adapted soil) while atrazine
mineralization remained slow in an adjacent soil under wheat that had never received atrazine (non-adapted soil). Carbon-14
atrazine degradation and formation of unextractable `bound' residues were determined during laboratory incubations in soil
alone or amended with di€erent organic amendments (OAs) and N sources. The OAs varied from readily biodegradable
(glucose) to more slowly mineralizable (cellulose and straw) and humi®ed organic matter (compost). The N forms included
mineral (NH4NO3, (NH4)2SO4 and Ca(NO3)2) and organic forms (adenine, arginine, albumin, biuret and pyrazine) which varied
in N availability. In the adapted soil, OA addition had little e€ects on atrazine degradation, whereas in the non-adapted soil, it
stimulated atrazine dealkylation more than triazine ring mineralization which always remained lower than in the adapted soil. In
both soils, mineral N decreased triazine ring mineralization. The depressive e€ect of the organic N forms on atrazine
mineralization increased with their N mineralization rate. Despite its slow N mineralization rate, the addition of biuret greatly

decreased atrazine mineralization, possibly because it is one of the last intermediates in atrazine degradation. The proportion of
bound residues increased with the total microbial activity after addition of OAs or organic N forms. In conclusion, rapid
triazine ring mineralization was dependent on micro¯ora adaptation after repeated atrazine application and was mainly
regulated by N availability in soil. # 2000 Elsevier Science Ltd. All rights reserved.

1. Introduction
Microbial degradation is the principal mechanism of
atrazine [6-chloro-N 2-ethyl-N 4-isopropyl-1,3,5-triazine2,4-diamine] dissipation from the environment (Esser
et al., 1975). Although this herbicide has been termed
as recalcitrant (Kaufman and Kearney, 1970), a large
variety of soil microorganisms are known to degrade
atrazine partially by N-dealkylation or dehalogenation
(Kaufman and Kearney, 1970; Behki and Khan, 1986;
Mougin et al., 1994; Bouquard et al., 1997). Complete
and rapid mineralization of the triazine ring has been

* Corresponding author. Tel.: +33-1-3081-5401; fax: +33-1-30815396.
E-mail address: houot@jouy.inra.fr (S. Houot).

reported (Mandelbaum et al., 1993; Assaf and Turco,

1994; Yanze-Kontchou and Gschwind, 1994; Mandelbaum et al., 1995; Radosevich et al., 1995) and the
possible adaptation of soil micro¯ora to atrazine
degradation after repeated ®eld applications has been
demonstrated (Barriuso and Houot, 1996; Vanderheyden et al., 1997). Microbial growth has been observed
with atrazine as sole C source (Behki and Khan, 1986;
Yanze-Kontchou and Gschwind, 1994; Stucki et al.,
1995). Rapid triazine ring mineralization however
seems to imply the development of microorganisms
using triazine nitrogen as a N source (Cook and HuÈtter, 1981; Mandelbaum et al., 1995; Radosevich et al.,
1995).
The addition of organic amendments (OAs) to soils
can modify the rate and pathways of pesticide degradation. Pesticides generally sorb readily to organic

0038-0717/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 6 7 - 4

390

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

Table 1
Main physicochemical characteristics of the soils

Continuous maize plot
Continuous wheat plot

Clay (g kgÿ1)

Silt (g kgÿ1)

Sand (g kgÿ1)

Organic C (g kgÿ1)

Total N (g kgÿ1)

CaCO3 (g kgÿ1)

pH (water)

253
241

623
445

91
171

11.5
17.4

1.2
1.7

33
61

8.2
8.0

matter. Thus OAs promoting sorption reduce pesticide
bioavailability and slow their biodegradation (Barriuso
et al., 1997). On the other hand, OAs may accelerate
pesticide degradation through a general stimulation of
the microbial biomass (Hance, 1973). Nitrogen availability in¯uences atrazine behaviour in soils also and
the e€ect varies with the form and amount of N present. A large concentration of mineral N greatly
decreased atrazine mineralization in soil when added
alone or with organic amendments (Entry et al., 1993;
Alvey and Crowley, 1995). On the other hand, dairy
manure including a large proportion of organic N
stimulated atrazine mineralization (Topp et al., 1996).
In this latter case, the greater availability of nutrients
allowed a larger number of atrazine degraders to multiply in the amended soil.
Our purpose was to test the e€ect of C and N availability on atrazine degradation in two soils: in one
soil, enhanced mineralization of atrazine was observed
and related to repeated ®eld applications of atrazine;
the other soil had similar physicochemical characteristics, but had never received atrazine under ®eld conditions and atrazine mineralization remained slow in
this soil (Barriuso and Houot, 1996). The dissipation
of 14C-atrazine via degradation and formation of unextractable, so-called `bound' residues was followed

during laboratory incubations in soils alone or
amended with di€erent OAs or N forms. The OAs and
the N compounds were selected according to their C
or N availability, respectively. Their connection to the

atrazine degradation pathway was another selecting
criteria for the N compounds.

2. Material and methods
2.1. Soils
Two adjacent experimental plots located in Grignon
(Yvelines, France) were sampled in the upper 20 cm in
April 96 and February 97, respectively for the two sets
of incubation. Both soils were Typic Eutrochrept
(Table 1). One plot has been cultivated with continuous maize since 1962 and has been treated yearly with
atrazine at recommended agronomic doses. The other
plot has been under continuous wheat since 1965 and
has never received atrazine. Soils were immediately
sieved (5 mm) after sampling and used for experiments
without storage.

2.2. Chemicals, organic amendments and N forms
Analytical standards of atrazine and its metabolites
(hydroxyatrazine, deethylatrazine, deisopropylatrazine,
deethyl-deisopropylatrazine) were purchased from
ChemService (West Chester, PA, USA). The [U-ring-14C]atrazine (speci®c activity 659 MBq mmolÿ1; radiopurity greater than 97%) was purchased from
Amersham (Buckinghamshire, UK).
Four OAs were compared (Table 2): glucose, cellu-

Table 2
Organic amendments (OAs) and N compounds: C and N contents and amount added gÿ1 of soil, calculated to approximately double the C content of the soil and to bring 2.5 mg of N gÿ1 of soil in all the treatments, respectively
OAs and N sources

C (mg kgÿ1)

N (mg kgÿ1)

Added C (mg kgÿ1)

Glucose
Cellulose

Straw
Compost
Ammonium nitrate
Calcium nitrate
Ammonium sulfate
Arginine
Albumin
Adenine
Biuret
Pyrazine

400.0
440.0
416.0
187.0
0.0
0.0
0.0
413.3
483.8

444.1
232.8
599.3

0.0
0.0
4.2
9.8
350.0
170.6
211.9
321.5
163.9
518.1
407.5
349.6

16.0
17.6
16.6

9.4
0.0
0.0
0.0
3.2
8.0
2.13
1.40
4.31

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

lose, wheat straw and a municipal solid waste compost. The cellulose was purchased from Sigma (St
Louis, MO, USA). The wheat straw came from the experimental farm of Grignon. It was ground in a plantblender to a 2 mm maximum particle size. The compost was obtained after 12 d of accelerated fermentation of municipal solid wastes and 6 weeks of
maturation. It was air dried and sieved (2 mm); the
smaller fraction was used.
Eight sources of N were used (Table 2): three mineral forms (NH4NO3, Ca(NO3)2 and (NH4)2SO4), an
amino-acid (arginine), a protein (albumin), the biuret
and two N-heterocycles, a purine (adenine) and a diazine (pyrazine).
2.3. Incubation experiments

Two sets of laboratory incubations of 14C-atrazine
with fresh soil samples equivalent to 10 g of dry soil
were conducted in triplicate and in hermetically stoppered jars at 28218C for 50 d.
In the ®rst incubation experiment, 14C-atrazine
degradation was followed in soil with or without addition of OA and with or without addition of mineral
N. Cellulose, straw and compost were added in powder and glucose and mineral N in solution. The
amount of OA was calculated in order to approximately double the organic C content of the soil in the
mixtures: 400 mg of straw and cellulose, 400 mg of
glucose (0.8 ml of a solution at 500 g lÿ1) and 500 mg
of compost (Table 2). Mineral N was added to half of
the jars corresponding to 25 mg of N (0.8 ml of a solution of NH4NO3 at 90 g lÿ1). Atrazine was added to
each jar to a concentration of 0.5 mg kgÿ1 of soil (0.8
ml of a solution of 14C-atrazine prepared in water at
6.25 mg lÿ1 and 9.94 MBq lÿ1), corresponding to the
dose applied in ®eld conditions. The water content of
the mixtures was adjusted to 85% of the soil ®eld capacity with MilliQ water (Millipore, Milford, MA,
USA) taking into account the water added with the
solutions of atrazine, glucose or mineral N. Similar incubations were carried out with 400 mg of cellulose or
straw and 500 mg of compost alone receiving the same
amount of atrazine and at a humidity equivalent to
85% of their water holding capacity.
In the second incubation experiment, 14C-atrazine
degradation was followed in soil with or without addition of the di€erent N-forms. In each treated jar, 0.6
ml of water solutions of either Ca(NO3)2 at 250 g lÿ1,
(NH4)2SO4 at 200 g lÿ1, arginine at 128 g lÿ1, albumin
at 277 g lÿ1 or pyrazine at 120 g lÿ1 was added, corresponding to 25 mg of N (Table 2). Adenine and biuret
were not soluble enough in water and 60 mg of biuret
and 48 mg of adenine were added as powder per jar
and mixed to soil. As previously, 14C-atrazine was
added to each jar in order to reach a concentration of

391

0.5 mg kgÿ1 of soil (0.5 ml of a solution of 14C-atrazine prepared in water at 10 mg lÿ1 and 19.4 MBq
lÿ1). The soil water content was adjusted to 85% of
the soil ®eld capacity with the atrazine and nitrogen
solutions supplemented with MilliQ water when
necessary.
The evolved 14C±CO2 and total C±CO2 were
trapped in 5 ml of 1 M NaOH placed into the incubation jars. The traps were sampled periodically and
replaced during the incubation. Total C±CO2 was analyzed by colorimetry on a continuous ¯ow analyser
(Skalar, Breda, the Netherlands) and 14C±CO2 was
determined by scintillation counting (Kontron Betamatic V; Kontron Ins., Montigny le Bretonneux,
France) using Pico¯uor scintillation cocktail (Packard,
Meriden, CT, USA). During the incubations with the
di€erent N forms, mineral N was extracted after 7, 14
and 50 days with 50 ml of 1 M KCl and analyzed by
colorimetry on the continuous ¯ow analyser.
2.4. Analysis of the

14

C-atrazine residues

At the end of the incubations, all the samples treated with 14C-atrazine were extracted by shaking in 50
ml of 10 mM CaCl2 in water for 16 h at 20 2 28C in
the dark. After centrifugation at 4000 g for 10 min,
the supernatant was removed and the residues
extracted again for 16 h by shaking with 50 ml of
methanol, three successive times. The extracted radioactivity was measured in the water and the methanol
extracts. The non-extractable radioactivity, corresponding to the `bound' residues was measured by
scintillation counting of the 14C±CO2 evolved after
combustion of the solid residues after methanol extraction (Sample Oxidizer 307, Packard, Meriden, CT,
USA). The water extracts were concentrated by solid±
liquid extraction with Lichrolut EN (200 mg) cartridges (Merck, Darmstadt, Germany). The cartridges
were eluted with 20 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 ®rst solvent used for the HPLC
analysis, 40/60 methanol/water (v/v) bu€ered with 50
mM ammonium acetate with pH adjusted to 7.4. The
three methanol extracts of the three replications were
pooled, then concentrated until dryness by evaporation
under vacuum; the residue was then dissolved in 2 ml
of the ®rst HPLC solvent. Samples for HPLC analyses
were ®ltered through a Cameo 13N syringe nylon ®lter
of 0.45 mm (MSI, Westboro, MA, USA). 14C-atrazine
and 14C-metabolites were analyzed by HPLC on a
Novapak C18 column (5 mm, 250 mm  4.6 mm;
Waters, Milford, MA, USA) with a Waters instrument
(600E Multisolvent Delivery System, 717 Autosampler)
equipped with a UV detector at 222 nm, coupled online with a radioactive ¯ow detector (Packard-Radio-

392

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

Fig. 1. E€ects of organic amendments and mineral N on total C±CO2 mineralization during incubation in soil from the continuous maize plots.
Results are expressed in g C±CO2 kgÿ1 soil. Standard deviations are indicated only where larger than symbols.

matic Flo-one A550). The mobile phase was methanol/
water bu€ered with 50 mM ammonium acetate with
pH adjusted to 7.4. The chromatography started with
40/60 methanol/water (v/v) for 15 min, then 80/20
methanol/water for 20 min. The mobile phase ¯ow
was 1.0 ml minÿ1 and the injected sample volume var-

ied from 300 to 800 ml, function of the total radioactivity detected in the extracts.
2.5. Sorption experiments
Ten millilitres of a solution of

14

C-atrazine at 10 mg

Fig. 2. Kinetics of total C±CO2 mineralization and total mineral N evolution during incubation with soil from the continuous maize, supplemented with di€erent N sources. Results are expressed in mg C±CO2 kgÿ1 soil and in mg N kgÿ1 soil, respectively. Standard deviations are
indicated where larger than symbols.

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

393

Fig. 3. Impact of organic amendments and mineral N on 14C-atrazine mineralization during incubation in soil from the continuous maize
(adapted soil) or wheat plot (non-adapted soil). Results are expressed as percent of the initial radioactivity. Standard deviations are indicated
only where larger than symbols.

lÿ1 in 10 mM CaCl2 and 0.11 MBq lÿ1 were added to
5 g of air-dried soil alone or soil-OA mixtures (in the
same proportions as in the incubation experiments:
200 mg straw or cellulose, 250 mg compost) into 25 ml
Corex glass centrifuge tubes with Te¯on caps. Sorption
experiments were also conducted with the OAs alone
(same amounts as previously). Triplicate samples were
carried out. After shaking for 24 h at 20 2 18C, the
samples were centrifuged at 7000 g for 20 min and
atrazine concentration in solutions (Ce, mg lÿ1) was
calculated from the supernatant radioactivity measurement. The amount of sorbed atrazine (S, mg kgÿ1)
was calculated from the di€erence of atrazine concentration in solution before and after sorption. The sorption coecient Kd (l kgÿ1) was calculated as
Kd ˆ S=Ce : The sorption coecient on an organic car-

bon
unit
basis,
Koc,
was
calculated
as
Koc ˆ 100  Kd =C, where C is the organic C content
(%).

3. Results and discussion
3.1. Total heterotrophic microbial activity
Total microbial activity was evaluated from the kinetics of total C mineralization. Both soils exhibited
similar heterotrophic activity and only the results of
the continuous maize plot are presented (Figs. 1 and
2). For both soils, 3 to 5% of the total organic C was
mineralized after 50 d of incubation (soil alone).
During incubations of the soil-OA mixtures, the

394

Adapted soil

Mineralized CO2
(% of initial 14C-atrazine)

Extractable residues
total
(% of initial

Without mineral N
Soil
Soil+straw
Soil+compost
Soil+cellulose
Soil+glucose

90.322.3
90.424.8
83.922.0
87.721.6
74.222.7

aa
a,b
b
a,b
d

With mineral N
Soil
Soil+straw
Soil+compost
Soil+cellulose
Soil+glucose

53.522.1
59.222.0
63.522.2
31.522.7
8.922.4

e
c
c
f
g

a
b

3.820.3
4.020.4
5.420.5
5.320.7
7.821.6
28.322.6
19.421.6
18.821.8
41.122.9
58.124.4

14

C-atrazine)

Bound residues
(% of initial 14C-atrazine)
atrazine
(% of initial

14

C-atrazine)

HYA
(% of initial

14

C-atrazine)

dealkylated
(% of initial

14

C-atrazine)

a
a
b
a,b
b

0.0b
1.3
0.0
0.0
0.0

0.3
0.0
0.2
0.5
2.8

3.5
2.7
5.1
4.7
4.9

6.720.1
7.020.5
10.520.6
6.920.2
9.520.1

a
a
b
a
b

d
c
c
e
f

0.0
0.8
0.2
0.7
0.0

2.5
0.0
0.2
0.0
12.3

25.8
18.6
18.3
40.3
45.1

17.720.4
19.821.5
20.020.7
25.720.2
30.620.4

c
c
c
d
e

Means within a column followed by the same letter do not di€er signi®cantly …P ˆ 0:05† according to Student's test.
Variance of HPLC analysis could not be given since the three replicates were combined before analysis.

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

Table 3
Distribution of the initial radioactivity from 14C-atrazine into the mineralized, extractable (water plus methanol extracts combined) and bound residue fractions after 50 d of incubation in the
adapted soil from the continuous maize plot receiving the di€erent OAs with or without mineral N. The percentages of initial radioactivity present as atrazine, hydroxyatrazine (HYA) and dealkylated metabolites (deethyl-atrazine, deisopropyl-atrazine and deethyl-deisopropyl-atrazine) in the extractable fraction are given

Non adapted soil

Mineralized CO2
(% of initial 14C-atrazine)

Extractable residues
total
(% of initial

Without mineral N
Soil
Soil+straw
Soil+compost
Soil+cellulose
Soil+glucose
With mineral N
Soil
Soil+straw
Soil+compost
Soil+cellulose
Soil+glucose
a
b

2.820.3
12.320.5
6.220.7
12.321.8
7.320.3

aa
b
c
b
c

0.420.1 d
3.320.2 a
0.920.1 e
1.520.1 f
0.220.01 d

14

C-atrazine)

Bound residues
(% of initial 14C-atrazine)
atrazine
(% of initial

14

C-atrazine)

HYA
(% of initial

14

C-atrazine)

dealkylated
(% of initial

14

C-atrazine)

65.321.9a
52.422.7b
49.523.1b
52.025.6 b
52.927.7 a,b

53.8b
15.0
11.9
14.1
34.2

1.6
1.7
2.7
1.2
1.0

10.0
35.6
34.9
36.7
17.6

32.621.3
38.421.5
47.921.0
36.820.6
38.321.4

a
b
c,d
b
b

69.524.6
49.422.4
56.626.3
56.723.0
54.024.8

60.7
1.4
15.7
1.7
12.1

2.8
0.5
2.5
1.0
2.8

5.7
47.2
38.2
53.9
39.2

33.521.5
50.220.9
47.721.6
45.821.0
45.720.6

a
c
c,d
d
d

a,c
b
a,b,c
b
b

Means within a column followed by the same letter do not di€er signi®cantly …P ˆ 0:05† according to Student's test.
Variance of HPLC analysis could not be given since the three replicates were combined before analysis.

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

Table 4
Distribution of the initial radioactivity from 14C-atrazine into the mineralized, extractable (water plus methanol extracts combined) and bound residue fractions after 50 d of incubation in the
non-adapted soil from the continuous wheat plot receiving the di€erent OAs with or without mineral N. The percentages of initial radioactivity present as atrazine, hydroxyatrazine (HYA) and
dealkylated metabolites (deethyl-atrazine, deisopropyl-atrazine and deethyl-deisopropyl-atrazine) in the extractable fraction are given

395

396

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

Fig. 4. Kinetics of 14C-atrazine mineralization during incubation with adapted and non-adapted soil, supplemented with di€erent N sources.
Results are expressed as percent of the initial radioactivity. Standard deviations are indicated where larger than symbols.

increased C mineralization compared to the soil alone
was attributed to the OA mineralization, assuming
that no interactions occurred between indigenous and
exogenous organic matter and corresponded to 2, 14,
22 and 44% of the initially added C for cellulose,
straw, compost and glucose, respectively. Mineral N
addition did not modify C mineralization in the soils
alone (Fig. 1). In contrast, the addition of mineral N
increased C mineralization for cellulose, straw and glucose, which reached 27, 32 and 61% of the initially
added C, respectively. Compost C mineralization was
not stimulated by mineral N addition.
Total C mineralization was not a€ected by
Ca(NO3)2 addition but was doubled when (NH4)2SO4
was added (Fig. 2). All the organic forms of N
enhanced C mineralization with di€erent intensities.
The mineralization of the added organic materials was
estimated from the increase of C±CO2 evolved during
the incubation as previously. At the end of the incubations, 8, 12, 49, 73 and 74% respectively of pyrazine,
biuret, adenine, arginine and albumin organic C were
mineralized.
ÿ
The evolution of mineral N (N±NH+
4 +N±NO3 )
was followed in the treatments with the di€erent Nforms. Again, the results were similar in the two soils
and only the results corresponding to the continuous
maize plot are presented (Fig. 2). At the end of the
control incubations, 1% of the soil organic N was
mineralized in both soils. All the N compounds
increased the mineral N concentration in soils, except

pyrazine and biuret which were the least biodegradable
organic materials. Mineral N remained constant when
Ca(NO3)2 or (NH4)2SO4 was added. As for C, N mineralization of the organic N compounds was estimated
from the increase in mineral N during the incubations
of amended soils as compared to the soils alone,
assuming that the addition of exogenous organic matter did not modify the indigenous N cycle. With adenine, N mineralization reached 26% of the added
amount and 45% with albumin and arginine and mineral N was mainly present as ammonium. With pyrazine and biuret, only 5% of the initial organic N was
mineralized at the end of the incubations, including as
much ammonium as nitrate. The availability of C and
N in the various organic N compounds increased in
the same order: pyrazine < biuret < adenineRalbumin ˆ
arginine:
3.2. Atrazine degradation in the control soils
During incubation with soil from the continuous
maize plot, atrazine mineralization increased rapidly
and reached a plateau corresponding to 90% of the initially added radioactivity after only 14 d (Fig. 3). At
the end of the incubation, 3.8% of the initially added
14
C remained extractable and 6.7% formed non-extractable bound residues (Table 3). No atrazine was found
in the extractable fraction which mainly included dealkylated metabolites (Table 3).
During incubation with soil from the continuous

14

C±CO2 (% of initial
C-atrazine)

Extractable residues

Bound residues (% of
initial 14C-atrazine)

14

14

total (% of initial
C-atrazine)

atrazine (% of initial
14
C-atrazine)

M16 (% of initial
14
C-atrazine)

HYA (% of initial
14
C-atrazine)

dealkylated (% of initial
14
C-atrazine)

Adapted soil
Control soil
Soil+pyrazine
Soil+albumin
Soil+adenine
Soil+arginine
Soil+(NH4)2SO4
Soil+biuret
Soil+Ca(NO3)2

93.020.6 aa
88.022.2 b
22.922.9 c
24.521.6 c
9.320.4 d
7.620.5 e
7.320.7 e,f
6.120.4 f

2.320.1 a
6.420.5 b
44.124.5 c
48.222.0 c
54.929.4 c,d
61.022.8 d,f
72.122.3 e
67.221.9 e,f

2.0b
2.2
28.0
38.3
43.6
54.0
48.8
57.2

0.0
0.3
1.7
1.4
1.8
1.6
0.1
0.0

0.0
0.1
0.7
3.9
1.3
0.7
6.4
4.3

1.3
3.8
16.7
4.1
3.6
4.5
16.8
5.6

6.620.1 a
9.120.4 b
29.520.1 c
29.320.4 c
35.620.6 d
31.220.1 e
21.820.2 f
26.420.1 g

Non adapted soil
Control soil
Soil+pyrazine
Soil+albumin
Soil+adenine
Soil+arginine
Soil+(NH4)2SO4
Soil+biuret
Soil+Ca(NO3)2

44.222.8 aa
19.421.7 b
0.320.04 c
0.720.2 d
0.120.0 e
0.220.0 f
0.620.1 d
0.120.0 e

25.623.5 a
34.9213.4 a,b
56.724.2 b,c
57.424.9 b,d
60.920.7 b,e
62.823.2 c,d,e
67.722.3 d
67.723.2 d

9.7
24.9
38.1
43.8
48.5
54.8
52.2
56.4

0.0
0.9
2.1
2.6
3.3
1.5
0.2
0.0

4.9
4.7
6.1
4.8
3.5
3.2
6.4
5.5

11.0
4.4
9.8
5.9
4.9
3.2
8.7
5.7

29.220.6
29.320.1
41.820.1
37.721.3
40.420.6
37.520.5
31.020.1
32.621.0

a
b

a
a
b
c,d
c
d
e
e

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

Table 5
Distribution of the initial radioactivity from 14C-atrazine into the mineralized (14C±CO2), extractable (water plus methanol extracts combined) and bound residue fractions after 50 d of incubation in the adapted and non-adapted soil receiving the di€erent N forms. The percentages of initial radioactivity present as atrazine, unidenti®ed metabolite (M16), hydroxyatrazine (HYA)
and dealkylated metabolites (deethyl-atrazine, deisopropyl-atrazine and deethyl-deisopropyl-atrazine) in the extractable fraction are given

Means within a column followed by the same letter do not di€er signi®cantly …P ˆ 0:05† according to Student's test.
Variance of HPLC analysis could not be given since the three replicates were combined before analysis.

397

398

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

wheat plot, atrazine mineralization increased progressively throughout all the incubation but remained very
low (Fig. 3). At the end of the incubation, 65.3% of
the initially added 14C remained extractable and 32.6%
formed bound residues (Table 4). Atrazine was the
dominant compound in the extractable fraction and
mainly dealkylated metabolites were detected with very
few hydroxyatrazine (Table 4).
These results con®rmed the presence of a microbial
community with the capacity to mineralize the triazine
ring of atrazine in the continuous maize plot (adapted
soil) but not in the continuous wheat plot (nonadapted soil) (Barriuso and Houot, 1996). Dealkylation of the atrazine amino-substituants has been
shown to be the major mechanism involved in atrazine
microbial degradation and was associated with low
rates of triazine ring degradation (Kaufman and Kearney, 1970). More recently, rapid atrazine degradation
and triazine ring mineralization have been demonstrated and dehalogenation has been shown to be the
®rst step of atrazine degradation in many studies with
a consortium of microorganisms or isolated strains
(Mandelbaum et al., 1993; Assaf and Turco, 1994;
Mandelbaum et al., 1995; Radosevich et al., 1995; de
Souza et al., 1998). Alternatively, dealkylation could
be the ®rst step of a rapid atrazine degradation and
dealkylation and dehalogenation can occur simultaneously (Yanze-Kontchou and Gschwind, 1994;
Shao et al., 1995; Stucki et al., 1995). Both hydroxyatrazine and dealkylated metabolites were detected in
the extracts after incubation in the adapted and nonadapted soils. However, microorganisms responsible
for triazine ring mineralization were active only in the
adapted soil. In the non-adapted soil, bound residue
formation constituted an alternative pathway of atrazine dissipation (Barriuso and Houot, 1996).
During the second set of experiments, similar results
were observed during atrazine incubation in the soil
from the continuous maize plot (Fig. 4), but atrazine
mineralization reached 44.2% in the control soil
sampled in the permanent wheat plot. This large and
unexpected atrazine percentage of mineralization was
probably related to the contamination of the wheat
plot by soil of the maize plot since both plots are adjacent.
3.3. Nitrogen e€ect on atrazine degradation
All of the N compounds decreased atrazine mineralization signi®cantly in both soils. In the adapted soil,
the intensity of the decrease varied with the form of N
added and atrazine mineralization was negatively correlated with the mineral N content of the soil in the
di€erent treatments at the end of the incubations
…r ˆ 0:797, P < 0.05). The largest decreases in atrazine
mineralization were observed with the mineral N

forms. With the same concentration of Ca(NO3)2,
Alvey and Crowley (1995) also observed a large inhibition of atrazine mineralization. With the organic N
compounds, atrazine mineralization decreased when N
availability of the molecules increased. Pyrazine was
the least available form of N; arginine, albumin and
adenine were the most easily degraded molecules (Fig.
2). The only exception was observed with biuret. In
spite of the low availability of biuret N, only 7.3% of
the initial atrazine was mineralized at the end of the
incubation. Biuret is one of the last metabolites in the
atrazine degradation pathway (Cook et al., 1985) and
large concentrations of this ®nal intermediate could
have inhibited the enzymatic transformations occurring
in atrazine degradation. Atrazine is used as an N
source by atrazine-degrading microorganisms (Mandelbaum et al., 1995; Radosevich et al., 1995) and mineral
N addition could inhibit atrazine mineralization by
o€ering an alternative source of N. However, since
large concentrations of mineral N are required to observe a negative e€ect on atrazine mineralization
(Alvey and Crowley, 1995), the synthesis or the activity of the enzymes responsible for triazine ring
degradation could also be a€ected (Entry et al., 1993).
Nitrogen has been found to regulate the synthesis or
the activity of enzymes like peroxidases (Li et al.,
1994; Kaal et al., 1995). The negative e€ect of N on
atrazine degradation was not related to the increase of
soil conductivity after addition of large salt concentration (Smith and Doran, 1996). Atrazine mineralization was not decreased when similar concentrations
(10 mM) of CaCl2 or Na2SO4 were added (results not
shown).
In the non-adapted soil, less than 1% of the initially
added atrazine was mineralized in all the N treatments,
except with pyrazine which had little e€ect on atrazine
mineralization as in the adapted soil.
In both soils, the largest residual extractable radioactivities were measured in the treatments which had
the largest depressive e€ect on atrazine mineralization:
the mineral N forms and the biuret (Table 5). The addition of N decreased triazine ring mineralization and
also atrazine partial degradation. Atrazine was always
the dominant compound in the extracts. With all the
N compounds, both hydroxyatrazine and dealkylated
metabolites were detected. An unidenti®ed metabolite
(M16) was detected with albumin, adenine, arginine
and (NH4)2SO4. It was chromatographied after 16 min
between hydroxyatrazine (13 min) and atrazine (21
min).
In both soils, the bound residue proportion
increased when atrazine mineralization decreased
(Table 5) and when the total microbial activity as estimated with the total C±CO2 evolved during the incubation, increased. This con®rmed the microbial

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401
Table 6
Distribution coecients Kd and Koc for atrazine sorption on organic
amendments, soils and soil-organic amendment mixtures

Organic amendment
Straw
Compost
Cellulose
Continuous maize plot
Soil alone
Soil+straw
Soil+compost
Soil+cellulose
Continuous wheat plot
Soil alone
Soil+straw
Soil+compost
Soil+cellulose

Kd (l kgÿ1)

Koc (l kgÿ1C)

15.0120.43 aa
10.1021.59 b
1.3220.12 c

36.121.0 a
54.228.5 b
3.020.3 c

0.5420.01
1.1620.05
0.9920.13
0.5820.05

d
c
c
d

47.120.7
42.921.7
49.826.4
20.621.7

b
a, b
b
d

0.7720.04
1.3320.03
1.1820.02
0.7820.04

e
c
c
e

44.222.2
40.821.0
46.520.7
23.121.1

b
a, b
b
d

a
Means within a column followed by the same letter do not di€er
signi®cantly …P ˆ 0:05† according to Student's test.

contribution to pesticide bound residue formation in
soils (Benoit and Barriuso, 1997).
3.4. Atrazine degradation in organic amended soils
Less than 1% of the initial radioactivity was mineralized when atrazine was incubated with compost, straw
or cellulose alone. The proportion of bound residues
was larger with compost (68.3% of the initial radioactivity) than with straw (28.0% of the initial radioactivity). Few bound residues were formed with cellulose
(0.6% of the initial radioactivity). Atrazine sorption
on straw was greater than on compost and was very
low on cellulose (Table 6). The larger anity of compost organic matter than straw organic matter for
atrazine, revealed by larger Koc values may partly
explain the larger proportion of bound residues formed
with compost. Sorption could be the initial process
leading to formation of highly stabilized unextractable
residues. With cellulose, most of the radioactivity
remained extractable (95.1% of the initial radioactivity) and mainly as parent atrazine. With compost,
44.3% of the initial radioactivity remained extractable
and atrazine was also the main component of the
extract. With straw, 75.5% of the initial radioactivity
was extracted after the incubation and only 23.8%
remained as atrazine. Mainly dealkylated metabolites
were detected (50.6% of the initial radioactivity).
The e€ect of adding OAs on atrazine degradation
di€ered in the adapted and non-adapted soils. In the
adapted soil, the addition of OAs had little e€ect on
atrazine mineralization which decreased signi®cantly
only when compost and glucose were added (Fig. 3).
With these OAs, bound residue formation slightly
increased (Table 3).

399

Straw and compost increased atrazine retention in
both soils, but cellulose did not (Table 6). Greater
atrazine sorption can decrease its availability for microbial degradation. This was observed in the adapted
soil for compost, con®rming the results of Barriuso et
al. (1997), while straw did not modify atrazine mineralization. The anity of straw organic C for atrazine
was lower than that of soil C, indicated by the smaller
Koc (Table 6). Thus, even if adsorbed on straw organic
matter, atrazine remained available for microbial
degradation in the adapted soil where the degrading
micro¯ora was very active.
Organic amendments can also increase pesticide
degradation by stimulating microbial activity (Entry
and Emmingham, 1995; Topp et al., 1996). Indeed, in
the non-adapted soil, all the OAs stimulated both total
microbial activity and atrazine mineralization. However both activities could not be directly related since
the di€erent OAs did not have the same e€ect on total
CO2 and 14CO2 evolution, con®rming the results of
Alvey and Crowley (1995). OA addition also increased
bound residue formation in the non-adapted soil and
the largest proportion of bound residues was observed
with compost (Table 4), probably because of its greater
anity for atrazine than the other OAs (Table 6).
In the adapted soil, the addition of OAs little modi®ed the nature of the extractable radioactivity with
dealkylated metabolites as dominant compounds
(Table 3). Atrazine was only detected after incubation
with straw and represented only 1.3% of the initial
radioactivity. The largest proportion of hydroxyatrazine was observed with glucose (36.5% of the extractable radioactivity). It could not be related to an
increase in chemical hydrolysis accompanying acidi®cation (Khan, 1978). The pH in the glucose-amended
soil incubation remained above 8.0 and did not
decrease as observed by Alvey and Crowley (1995).
In the non-adapted soil, OA addition increased the
partial degradation of atrazine. The extractable atrazine represented 53.8% of the initial radioactivity at
the end of the control incubation and only 34.2% of
the initial radioactivity when glucose was added and
12±15% with the other OAs (Table 4). Mainly dealkylated metabolites were detected in the extracted radioactivity with all of the OAs. The largest increases in
atrazine dealkylation and mineralization were observed
when straw or cellulose were added which could mean
that cellulolytic microorganisms could be active in
atrazine degradation.
When mineral N was added simultaneously with the
OAs, atrazine mineralization decreased signi®cantly as
compared to the same treatment without mineral N
(Fig. 3), con®rming the inhibiting e€ect of mineral N
on atrazine mineralization previously described. In the
adapted soil, the addition of OAs and mineral N differentiated the e€ects of the OAs (Table 3). Atrazine

400

R. Abdelha®d et al. / Soil Biology & Biochemistry 32 (2000) 389±401

mineralization was very low in the presence of glucose
(8.9%), reached 31.5% with cellulose, 59.2% with
straw and 63.5% with compost. Simultaneously, in all
the treatments, the bound residues increased signi®cantly with the total microbial activity as revealed by
the kinetics of total CO2 mineralization. This again
con®rmed the relation between bound residue formation and microbial activity (Benoit and Barriuso,
1997). The direct inhibitory e€ect of mineral N, as previously discussed, probably also decreased atrazine
mineralization. Few atrazine and mainly dealkylated
metabolites were detected in the extractable fractions
(Table 3). With glucose, a large proportion of hydroxyatrazine was observed.
In the non-adapted soil, the simultaneous addition
of mineral N and OAs decreased atrazine mineralization (less than 1% of the initial radioactivity was
mineralized with most of the OAs and 3% with straw)
but increased the partial degradation of atrazine. Dealkylated metabolites were the predominant compounds
in the extractable radioactivity (Table 4). Atrazine
stabilization as bound residues did not increase signi®cantly.
In summary, the e€ect of C and N availability on
atrazine degradation in soils varied in relation to the
micro¯ora active in atrazine degradation. In a soil
adapted to atrazine mineralization, the OAs alone had
little e€ect on atrazine degradation. In a non-adapted
soil, OA addition stimulated atrazine mineralization,
but mineralization was always lower than in the
adapted soil. However, large atrazine dealkylation was
observed which increased when mineral N was added.
In the non-adapted soil, the OAs promoted dealkylation but not mineralization. In both soils, mineral N
decreased atrazine mineralization. The negative e€ect
of organic N compounds increased with their N mineralization rate, thus their N availability. Both triazine
ring mineralization and atrazine dealkylation or hydroxylation were inhibited by N addition.

Acknowledgements
This work was ®nancially supported by the INRA
program ``AIP- Etude et Gestion de l'EcosysteÁme
Sol''. The authors thank J.N. Rampon and V. Bergheaud for their technical assistance.

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