Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue5.2000:

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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. UniteÂ de Science du Sol, BP 01, 78850 Thiverval-Grignon, France b

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

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

[U-ring-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 applied14C-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 initial14C-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

napropamide (Walker and Welch, 1992) has been documented. Atrazine [6-chloro-N2-ethyl-N 4-isopro-pyl-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 cata-lyzed by bacteria or fungi was the classical pathway of microbial degradation described (Kaufman and Kear-ney, 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 bac-terial 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

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* Corresponding author. Tel.: 5401; fax: +33-1-3081-5396.

E-mail address:houot@jouy.inra.fr (S. Houot). 1

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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 (%) Texture C

(g kgÿ1) N (g kgÿ1)

C/N (ratio)

pH (H2O)

CaCO3

(g kgÿ1) CEC (cmol+kgÿ1)

clay silt sand

French soils

Plots frequently receiving atrazine (continuous maize or rotation including maize)

Citeaux R M 18.0 50.0 30.9 L 14.0 1.43 9.8 7.9 11 11.0

Baccon R M 37.1 57.1 5.7 SC 15.6 1.70 9.2 7.2 0 23.4

Montoison C M 20.6 17.9 41.4 SCL 15.9 1.83 8.7 8.2 195 14.5

CoÃte Saint AndreÂ C M 18.2 38.7 43.0 L 13.8 1.30 10.6 7.0 0 9.1

Chigy C M 41.2 51.0 5.8 SC 64.5 6.52 9.9 7.8 19 38.0

Chabeuil C M 15.8 36.1 48.0 L 17.2 1.37 12.6 7.1 0 9.1

Le Rheu R M 16.6 70.7 12.6 SL 11.4 1.24 9.2 6.2 0 7.3

Loustalet C M 19.2 65.7 15.0 SL 17.7 1.93 9.2 5.6 0 9.7

Plumelec R M 16.1 48.2 35.6 L 29.4 2.59 11.4 5.7 0 13.0

Salinis C M 25.3 57.0 17.6 SL 51.1 3.44 14.9 5.5 0 15.3

Grignon 1: DeheÂrain C M 20.3 61.3 14.8 SL 11.3 1.20 9.4 8.2 35 15.6

Grignon 2: Plateau R M 22.5 60.2 15.3 SL 15.2 1.45 10.5 8.0 19 16.9

Grignon 3: Plateau R W 22.3 61.1 14.7 SL 14.9 1.42 10.5 8.0 18 17.2

Grignon 4: Plateau C M 22.1 64.6 10.6 SL 13.0 1.24 10.5 8.1 26 16.5

Grignon 5: Block 1 untreated C M 20.1 75.0 4.3 SL 11.3 1.23 9.2 8.0 6 12.7

Grignon 6: Block 1 sludge C M 16.8 77.8 4.8 Sl 11.9 1.31 9.1 7.9 6 13.1

Grignon 10: Block 2 mineral N C M 15.9 79.8 4.1 SL 12.1 1.21 10.0 6.4 0 12.2

Grignon 11: Block 2 sludge C M 17.0 78.7 4.1 SL 12.7 1.27 10.0 6.6 0 12.0

Plots never receiving atrazine

Grignon 16: plateau C W 21.0 59.1 16.5 SL 18.3 1.70 10.8 8.0 33 17.7

Boyer C G 39.5 26.4 15.8 CL 39.1 4.32 9.03 8.0 174 24.3

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 6.3 CL 20.2 2.36 8.56 8.1 83 20.0

Boyer: maize for 3 yr C M 44.2 39.8 5.6 SC 32.1 3.85 8.34 8.0 99 25.4

Boyer: maize for 2 yr C M 32.7 56.8 7.2 SC 28.2 3.19 8.84 7.9 32 19.6

Canadian soils

Harrow 1 R M 47.3 24.2 28.5 C 21.7 ndb _nd _6.2 ₀ _17.8

Harrow 2 R M idc _id _id _id _22.8 _nd _nd _6.6 ₀ _18.1

Harrow 3 C M id id id id 20.2 nd nd 6.1 0 16.5

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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 ap-plication 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 fre-quently treated with atrazine under ®eld conditions.

The present work was undertaken to broaden the type of soils tested for accelerated degradation of atra-zine, 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 herbi-cide 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 exper-iments 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 maize-wheat 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 ex-periment studying the eect of sewage sludge appli-cation 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 tinuous grass pasture, are progressively being con-verted to continuous maize, providing a range of well-characterized 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

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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 con-ditions.

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 pre-vious 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 pro-vided 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 or-ganic carbon was determined by sulfochromic oxi-dation 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 cat-ion 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 lÿ1and 8.8 MBq lÿ1and solution B in 10 mM CaCl2at 9.85 mg lÿ1and 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 ofÿ104Pa, 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 kgÿ1soil. The jars each received a CO2trap

con-sisting of a vial containing 5 ml of 0.5 M NaOH and

were hermetically sealed and incubated statically at 28 218C 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 lumines-cence extinction, with a scintillation counter Betamatic V (Kontron Ins., Montigny le Bretonneux, France).

2.4. Analysis of the distribution of14C-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 metha-nol extracts was determined by liquid scintillation counting. The non-extractable radioactivity, corre-sponding 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 concen-trated 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 vac-uum 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 14 C-metabolites 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 (Pack-ard-Radiomatic Flo-one A550). The mobile phase was methanol±water buered with 50 mM ammonium acetate with pH adjusted to 7.4. The gradient chroma-tography 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 gradi-ent (gradigradi-ent 6 in Waters software) after 21 min and remained constant until 35 min. The mobile phase ¯ow was 1.0 ml minÿ1and the injection volume was 800ml.

S. Houot et al. / Soil Biology & Biochemistry 32 (2000) 615±625

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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 deter-mined 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 ana-lyzer (Skalar, the Netherlands). The nitrogen minera-lized 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 sol-ution B of atrazine in 25 ml Corex centrifuge tubes with Te¯on caps. The suspensions were agitated for 24 h at 20218C. 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 scintil-lation liquid with the scintilscintil-lation counter. The amount of adsorbed atrazine was calculated from the dierence between the initial herbicide concentration and the equilibrium concentration. TheKdsorption coecients were calculated as the ratio of the amount of adsorbed herbicide (mg) per unit mass of soil (kg) to the equili-brium concentration (mg lÿ1). The sorption coecients relative to the soil organic carbon (Koc) were obtained by dividing the Kd values by the organic carbon con-tent 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 coe-cients and the variables describing global microbial ac-tivity. 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 mineraliz-ation in relmineraliz-ation with the soil micro¯ora acclimatiz-ation; (2) we wanted to determine if accelerated mineralization of atrazine was related to soil physico-chemical 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).

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 phy-sico-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 in-dicators of the total microbial activity in the soils (Table 2). In most soils, the size of the microbial bio-mass corresponded to about 2% of the total organic carbon, a proportion frequently encountered (Jenkin-son 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 sig-ni®cantly with clay content r0:691, P < 0.01; Table 3), in agreement with the results of Chaussod et al. (1986). There was also a signi®cant but weaker re-lationship of microbial biomass with total soil organic carbon of the soilsr0:403, P< 0.05; Table 3). Car-bon mineralization was not correlated with the soil or-ganic carbonr0:131; Table 3) but N mineralization was r0:454, P< 0.01). Both C and N mineraliz-ation increased signi®cantly with soil clay content (r= 0.730 and 0.413, respectively for C and N mineraliz-ation). On the other hand, soil pH was correlated posi-tively with C mineralization r0:661, P< 0.01) but negatively correlated with N mineralization

r ÿ0:483,P<0.01).

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Table 2

Microbial biomass measured in fresh soils, total carbon and total nitrogen mineralized in soils at the end of the atrazine mineralization incu-bation (all expressed on a dry weight basis). Sorption coecients expressed on a total dry weight (Kd) or total organic carbon (Koc) basis and fraction of initial14_{C-atrazine mineralized during the 35 d incubations}

Soils Microbial biomass

(mg C kgÿ1 soil)

Cmin (mg C kgÿ1

soil) Nmin (mg N kgÿ1

soil)

Kd (l kgÿ1

soil)

Koc (l kgÿ1

C)

Atrazinemin (% initial14C)

French soils

Citeaux 309214 506248 27.722.1 0.8620.01 6121 84.129.0

Baccon 320211 26627 9.124.2 1.0420.04 6622 70.421.2

Montoison 31324 395214 12.020.6 0.8820.04 5523 68.021.7

CoÃte Saint AndreÂ 231217 382214 15.320.6 0.8520.08 6226 67.822.7

Chigy 480217 601246 21.121.1 4.1320.04 6421 66.522.2

Chabeuil 18929 353217 14.122.5 1.1020.01 6421 61.621.3

Le Rheu 204211 26525 22.924.4 0.6920.02 6022 23.322.3

Loustalet 23524 261211 28.4212.4 1.2320.06 6923 19.920.9

Plumelec 20927 371246 26.823.7 2.5520.29 87210 10.920.3

Salinis 137245 27724 33.423.1 3.9420.16 7723 8.520.3

Grignon 1: DeheÂrain 22924 336217 5.921.9 0.5920.01 5221 72.021.6 Grignon 2: Plateau 335219 461215 12.623.1 0.8120.01 5321 72.120.6 Grignon 3: Plateau 329246 430243 8.522.1 0.7520.06 5024 67.921.8 Grignon 4: Plateau 25023 33823 2.923.8 0.6020.04 4623 71.722.0 Grignon 5: Block 1 untreated 24125 317224 12.321.3 0.7220.03 6423 80.120.6 Grignon 6: Block 1 sludge 255210 361225 14.220.0 0.7620.09 6428 77.321.9 Grignon 7: Block 2 untreated 21628 26828 7.320.9 0.9420.06 7925 74.021.4 Grignon 8: Block 2 untreated 18325 28823 15.221.7 1.0820.02 8222 76.821.1 Grignon 9: Block 2 untreated 15222 23528 7.520.6 0.9720.00 9020 23.620.9 Grignon 10: Block 2 mineral N 18328 224211 5.921.2 0.9520.02 7922 13.821.0 Grignon 11: Block 2 sludge 147212 270225 14.422.8 0.9220.02 7222 58.323.3 Grignon 12: Block 3 untreated 11629 252227 7.321.4 0.9220.08 9428 20.221.0 Grignon 13: Block 3 sludge 164215 241213 8.721.2 0.9720.10 8329 11.020.4 Grignon 14: Block 4 untreated 17525 243213 12.420.4 0.8120.06 7125 65.022.5 Grignon 15: Block 4 sludge 193210 27028 15.422.8 1.0720.07 8425 35.620.6 Plots never receiving atrazine

Grignon 16: Plateau 39925 433250 15.120.3 0.9420.06 5123 13.420.2 Boyer: Permanent grass 167327 1049213 44.620.6 2.0120.03 5221 16.220.3 Plots under continuous maize since various length of time and on the same type of soil

Boyer: maize for 21 yr 502215 516245 18.324.6 0.9420.00 4720 71.722.8 Boyer: maize for 8 yr 813222 53126 22.822.1 1.2120.01 4720 68.821.2 Boyer: maize for 5 yr 54128 552223 20.821.8 1.0420.07 4623 65.822.0 Boyer: maize for 3 yr 918245 91827 55.222.1 1.3820.00 4320 66.122.3 Boyer: maize for 2 yr 1138215 856220 27.920.4 1.4920.11 5324 62.320.8

Canadian soils

Harrow 1 615238 20224 31.629.2 1.4820.19 6829 3.5a

Harrow 2 821260 17526 22.6217.8 1.4520.13 6326 5.7

Harrow 3 38523 16527 35.5214.5 1.5020.07 7424 4.8

Harrow 4 644289 8323 19.821.9 1.2420.01 7521 11.0

Harrow 5 40228 14925 32.026.5 1.5820.08 8624 11.1

Harrow 6 473277 13527 11.026.8 1.4620.06 8024 16.7

Harrow 7 47321 183211 19.024.5 1.5620.03 7121 30.8

Harrow 8 41322 12925 12.2211.4 1.4620.01 7420 48.1

Harrow 9 427273 14225 25.725.8 1.5220.04 7622 23.8

Winchester 1 631210 169220 22.426.4 1.2520.02 5521 42.5

Winchester 2 66926 13826 10.725.8 1.7820.04 4721 40.5

Winchester 3 585216 163213 25.622.0 1.4720.05 5122 52.5

Winchester 4 71729 192236 27.225.1 1.6820.02 5121 53.3

Plots with last atrazine application in 1990

Winchester 5 740223 399212 56.525.5 1.7020.00 7420 1.9

Winchester 6 602214 21828 27.823.5 1.1920.12 5426 3.7

a_{Only one replicate was realized to study atrazine mineralization in the Canadian soils.}

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3.3. Atrazine retention on soils

For most of the soils, the sorption coecient (Kd) varied within the reference values of 0.2±2.46 l kgÿ1 (Tomlin, 1994), but was larger in the most organic soils sampled at Chigy and Salinis (Table 2). Kd was positively correlated with the carbon organic content of the soils r0:936, signi®cant at the 1% level; Table 3) con®rming numerous published results (Has-sett and Banwart, 1989; Barriuso and Calvet, 1992; Weber et al., 1993). On the other hand,Kocvalues var-ied from 47 to 87 l kgÿ1 C, in the range of the refer-ence data (39±155 l kgÿ1 C) reported in the Pesticide Manual (Tomlin, 1994). Koc signi®cantly increased when soil pH decreased r ÿ0:834, P < 0.01; Table 3). The quality of the organic matter in soils with dierent pH could contribute to this pH eect (Paya-Perez et al., 1992; Seybold et al., 1994) and sorp-tion has been shown to be greater on acidic than on organic matter with neutral pH values (Weber et al., 1993).

In the Boyer soils, the sorption coecients Kd

tended to decrease with the organic matter content of the soils as the duration of continuous maize cropping increased. The normalized sorption coecient Koc

remained rather constant (average of 48 l kgÿ1 C) in the six plots and the quantity of organic matter was the main factor of variation in atrazine sorption.

3.4. Atrazine mineralization and relationship with soil physico-chemical characteristics

Representative kinetics of atrazine mineralization are presented in Fig. 1 and the fraction of atrazine mineralized at the end of the incubations is found in Table 2. There was a clear eect of atrazine treatment history on atrazine mineralization. Four soils either had never been treated with atrazine in ®eld conditions

(2 French soils), or had been treated 6 yr prior to sampling (2 Canadian soils). In all of these soils with no history of atrazine treatment, atrazine mineraliz-ation was slow and by the end of the incubmineraliz-ation only 20% or less of the initially applied atrazine was miner-alized. This behaviour was in sharp contrast with that of most of the soils which had been treated with zine in the ®eld. In the majority of French soils atra-zine mineralization started without a lag, was rapid and after 20 d of incubation, reached a plateau corre-sponding to 60 to 80% of the initially added atrazine. Such high rates of atrazine mineralization are charac-terisic of enrichment cultures growing at the expense of atrazine (Mandelbaum et al., 1995; Radosevitch et al., 1995; Topp et al., 1995; Topp et al., 1996). Similar results have been observed in Grignon soils (Barriuso and Houot, 1996). Some of the soils obtained from Table 3

Correlation matrix obtained with the fraction of atrazine mineralized after a 35 d incubation in the French and Canadian soils (``Atrazinemin'' as percentage of the initial added amount), the physico-chemical characteristics of the soils (organic C and clay content, pH), the sorption coe-cientsKdandKoccharacterizing atrazine retention in the soils and variables describing the total microbial activity in the soils, the microbial bio-mass (Biobio-mass), the Carbon and Nitrogen mineralized after 35 d of incubation (Cminand Nmin). The data from the French and Canadian soils frequently treated with atrazine and Boyer 21 were used for all the correlations (total of 39 soils). The correlations were signi®cant for coecients larger than 0.325 (P< 0.05,) and highly signi®cant for coecients larger than 0.418 (P< 0.01,)

Atrazinemin Organic C Clay pH Kd Koc Biomass Cmin Nmin

Atrazinemin 1.000

Organic C ÿ0.150 1.000

Clay 0.398 _0.331 _1.000

pH 0.830 _ÿ_0.207 _0.206 _1.000

Kd ÿ0.325 0.936 0.342 ÿ0.367 1.000

Koc ÿ0.538 ÿ0.157 ÿ0.027 ÿ0.550 0.163 1.000

Biomass ÿ0.188 0.403 _0.691 _ÿ_0.064 _0.217 _ÿ_0.420 _1.000

Cmin 0.614 0.131 0.730 0.661 0.060 ÿ0.334 ÿ0.333 1.000

Nmin ÿ0.432 0.454 0.413 ÿ0.483 0.505 0.055 0.367 ÿ0.081 1.000

Fig. 1. Representative kinetics of [U-ring-14_{C]atrazine mineralization} during laboratory incubations in French and Canadian soils. Results are expressed in 14_C-CO

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Canada likewise rapidly mineralized the herbicide. In these soils atrazine mineralization occurred after a lag phase of 5 d and reached a total of 55% of the initially applied atrazine by the end of the incubation (Fig. 1).

Surprisingly, some soils which had been frequently treated with atrazine in the ®eld did not mineralize this herbicide rapidly. These soils accumulated 14CO2

rather slowly and linearly to a total of 20% or less of the initially applied atrazine by the end of the incu-bation (Fig. 1). This was observed both in some French and in some Canadian soils. Among the fre-quently-treated soils, the fraction of atrazine minera-lized during the incubation was compared with a variety of soil physical, chemical and biological prop-erties to see if any of these could explain the propen-sity to mineralize atrazine (Table 3). The fraction of mineralized atrazine was strongly correlated with soil pH r0:830, P< 0.01, Table 3). The in¯uence of soil pH on microbial adaptation to pesticide degra-dation has been shown to be non-speci®c for other pesticides (Suett et al., 1996b). The correlation between atrazine mineralization and soil pH is in agreement with previous observations suggesting that atrazine is mineralized more slowly in acidic than in alkaline soils, all frequently exposed to atrazine under ®eld conditions (Pussemier et al., 1997). A plot of pH ver-sus the fraction of atrazine mineralized yielded a sig-moidal curve (Fig. 2). As a matter of fact, linear regression reported in the correlation matrix poorly described this sigmoidal curve. In soils with pH values larger than 6.5, accelerated mineralization of atrazine was observed with a maximum accumulated 14CO2

varying from 50 to 80% of the initially added atrazine. However, when the soil pH value was lower than 6.0,

14

CO2 accumulation remained below 25% of the

in-itially added atrazine. When the soil pH values was between 6.0 and 6.5, atrazine mineralization increased rapidly with soil pH. Two anomalous soils did not conform with this relationship: Harrow 2 and 4. Although they had relatively neutral pH values of 6.6 and 7.0, accumulated 14CO2 reached only 4.7 and

11.0% of the initially added atrazine (Table 2). The most notable feature of these soils is their high clay content, which may prevent accelerated mineralization of atrazine (Schiavon, pers. comm.).

Bioavailability and consequently biodegradation, of organic substrates can be limited by sorptive retention to soil (Scow and Hutson, 1992). In our case, atrazine mineralization was not signi®cantly correlated with Kd

values describing its retention to soils r ÿ0:325). There was no clear relationship between atrazine min-eralization and measurements of ``total'' soil microbial activity. Atrazine mineralization was not correlated with the size of the microbial biomass, but positively and signi®cantly correlated with C mineralization. Both Koc and Cmin could be related to the nature of

soil organic matter. Atrazine retention relative to the total organic carbon (Koc) decreased when C easily mineralizable increased, maybe due to the low atrazine retention on easily mineralizable organic matter. On the other hand, atrazine mineralization was negatively correlated with N mineralization r ÿ0:432, P < 0.01). High concentrations of mineral N in soil have been shown to inhibit atrazine mineralization (Alvey and Crowley, 1995).

3.5. Fate of atrazine residues in soils

The distribution of the radioactivity between miner-alized, extractable and non-extractable fractions was determined in some of the French soils at the end of the incubations (Table 4). In the soils showing acceler-ated degradation, the residual radioactivity was ap-proximately equally partitioned between the extractable and non-extractable fractions, both repre-senting 5 to 10% of the initial 14C-atrazine. In ad-dition to atrazine, four metabolites were detected by HPLC-RD in the extracts, corresponding in retention time to three dealkylated metabolites and hydroxyatra-zine (Fig. 3). The metabolites represented more than 50% of the extractable radioactivity in the soils with the highest rates of atrazine mineralization.

In the non-degrading soils, an average of 50% of the initial radioactivity remained extractable with 30% of non-extractable bound residues formed (Table 4). The quantity and the proportion of atrazine in the extracts increased with the amount of extractable Fig. 2. Relationship between the fraction of mineralized atrazine (as

percent of initial14_{C added) at the end of the incubations and soil} pH in French and Canadian soils. Only the soils frequently receiving atrazine are represented (39 soils as in correlation study).

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Table 4

Distribution of the initial radioactivity from 14C-atrazine between the mineralized, extractable (water+methanol extracts) and bound residue fractions after 35 d of incubation in some of the French soils

Soils Mineralized (% initial added14C) Extractable (% initial added14C) Bound residues (% initial added14C)

Citeaux 84.129.0 6.320.4 6.720.3

Baccon 70.421.2 4.620.5 7.720.2

Montoison 68.021.7 7.620.3 7.820.1

CoÃte Saint AndreÂ 67.822.7 6.820.4 10.020.3

Chigy 66.522.2 7.921.1 6.121.2

Chabeuil 61.621.3 13.420.3 10.120.3

Le Rheu 23.322.3 46.225.3 24.621.7

Loustalet 19.920.9 42.921.3 30.221.0

Plumelec 10.920.3 53.921.3 30.322.1

Salinis 8.520.3 57.921.2 30.720.4

Grignon 1 72.021.6 4.920.4 6.320.2

Grignon 2 72.120.6 6.820.3 8.120.2

Grignon 3 67.921.8 7.920.7 9.120.4

Grignon 4 71.722.0 5.320.5 6.620.3

Plots never receiving atrazine

Grignon 16 13.420.2 57.221.1 25.320.2

Boyer: Permanent grass 16.220.3 48.122.9 28.920.5

Plots under continuous maize since various length of time and on the same type of soil

Boyer: maize for 21 yr 71.722.8 6.620.2 7.120.1

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radioactivity at the end of the incubation. Accumu-lation of hydroxyatrazine was observed.

In the metabolic pathway of atrazine degradation, dealkylation of the two alkylamino groups used to be considered as the dominant route of atrazine trans-formation (Cook, 1987), associated to slow atrazine mineralization. More recently, microbially-catalyzed hydrolytic dehalogenation has been demonstrated to be the ®rst step of atrazine degradation in the soils, followed by the rapid mineralization of the molecule (Mandelbaum et al., 1993). Only transient accumu-lation of hydroxyatrazine has been observed (Mandel-baum et al., 1995; Topp et al., 1996). Among the soils studied in the present work, rapid atrazine mineraliz-ation was observed in the so-called adapted soils, with-out accumulation of metabolites. In the acidic soils, slow atrazine mineralization was associated with metabolites accumulation. Both hydroxyatrazine and dealkylated metabolites were identi®ed suggesting that atrazine degradation could be initiated by both dehalo-genation or dealkylation, but stopped after the ®rst step of degradation in these apparently non-adapted soils. Abiotic hydrolysis of atrazine could also partici-pate in hydroxyatrazine accumulation in the acidic soils. This increase of clay catalyzed hydrolysis of atra-zine to hydroxyatraatra-zine at low pH and the stronger sorption of hydroxyatrazine at lower pH could partly explain the decreased mineralization of atrazine at low pH (Laird, 1996). In these acidic soils, even if atrazine was degraded to hydroxyatrazine, the low bioavailabil-ity of hydroxyatrazine could result in low mineraliz-ation.

3.6. Atrazine mineralization in relation to the number of ®eld applications

The rate of atrazine mineralization tended to

increase with the duration of the continuous maize and the consequent increasing successive number of atrazine applications (Fig. 4). The more frequent the application of atrazine, the higher the initial rate of mineralization and the larger the total amount of

14

CO2 accumulated by the end of the incubation. As

few as two successive annual atrazine applications accelerated degradation when compared to soil taken from an adjacent untreated sodded ®eld with otherwise comparable properties.

In summary, accelerated degradation of atrazine has been observed in many French and Canadian soils fre-quently treated with atrazine in ®eld conditions. The soil pH seemed to be the most important soil charac-teristics related to atrazine degradation, with signi®-cant mineralization in soils with pH values in excess of 6.5. However, other soil properties may intervene since two soils with large clay contents mineralized little atrazine, in spite of having pH values higher than 6.5. Atrazine retention in soil did not in¯uence its mineral-ization rate. Soils that mineralized little atrazine gener-ally promoted the formation of large amounts of bound residues. Accelerated degradation of atrazine was observed in soils that received as few as two suc-cessive applications in the ®eld. The isolation and characterization of atrazine-degrading microorganisms in some of these soils is currently being undertaken.

Acknowledgements

The authors thank the following persons for provid-ing soil samples: E.G. Gregorich, C. Drury, A. Villard, S. Nicolier from AGPM (French association of maize producers) and her local collaborators. We thank V. Bergheaud, J.-N. Rampon and H. Bork for technical assistance. Work in E. Topp's laboratory was in part funded through an agreement with Novartis Crop Pro-tection.

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