Rapid meneralization of the s-triazine ring of atrazine in soils in relation to soil management

PII:

Soil Biol, Biochem. Vol. 28, No. lO/ll, pp. 1341-1348, 1996 Copyright 0 19% Elswier science Ltd Printed in Great Britain. All rights reserved solI3&0717(%)80144-7 0038-0717/% S15.00 + 0.00

RAPID MINERALIZATION OF THE S-TRIAZINE RING OF ATRAZINE IN SOILS IN RELATION TO SOIL MANAGEMENT E. BARRIUSO*

and S. HOUOT

LJnitk de Science du Sol, Institut National de la Recherche Agronomique, I.N.R.A.,

78850, Thiverval-Grignon, (Accepted

France

31 May 1996)

Smnrnary-Mineralization of the triaxine ring of atraxine was studied in soils with similar physicochemical properties, from experimental plots under different crop rotations located in Grignon, France Rapid mineralization rates were found in plots under continuous maize receiving atraxine every year. On the contrary, low mineralization rates were measured in plots under continuous wheat or permanent grass that had never received atraxine. The rapid mineralization of the atraxine-ring was observed without any previous laboratory microbial enrichment. It was also related to the presence of a chloro-substituent on the ring: rapid mineralization was also observed with simaxine, another chloro-s-triaxine, but not with terbutryn, a thiomethyltriaxine. The characterization of the extractable metabolites during the incubation experiments did not allow determination of the degradation pathway. In these soils, two competitive dissipation processes are proposed: (1) rapid dissipation through ring cleavage and mineralization in soil from the plot receiving atrazine every year, (2) more progressive dissipation through formation of bound residues in the other soils. The rate of mineralization of the atraxine-ring varied during the year and was rather sensitive to soil storage conditions. Copyright 0 1996 Elsevier Science Ltd

INTRODUCTION

Atrazine [6chloro-N’-ethyl-fl-isopropyl-1,3,5-triazine-2,4diamine] is a herbicide widely used to control numerous broad-leaved weeds. It is quite mobile in soil and has been frequently detected in ground and surface waters. Although atrazine and other s-triazines have often been termed recalcitrant (Kaufman and Keamey, 1970), biodegradation remains the principal process of atrazine dissipation in soils. A large variety of soil microorganisms are able to degrade atrazine partially by N-dealkylation or dehalogenation reactions (Kaufman and Keamey, 1970; Behki and Khan, 1986; Mougin et al., 1994). Soil incubation experiments with selective inhibition of bacteria or fungi suggested that both were necessary for triazine-ring mineralization into CO* in an agricultural soil (Levanon, 1993). Many fungi are able to N-dealkylate atrazine, but cannot degrade the ring (Kaufman and Blake, 1970; Behki and Khan, 1986; Hickey et al., 1994). Fungal transformation products of atrazine could be precursors for the bacterial mineralization of the triazine-ring (Levanon, 1993). Recently, complete mineralization has been reported for a bacterial consortium (Gschwind, 1992; Mandelbaum et al., 1993a) and *Author for correspondence.

for pure bacterial isolates (Mandelbaum et al., 1995; Radosevich et al., 1995). Use of carbon from the ring substituents has been reported and microbial growth measured with atrazine as sole carbon source (Yanze-Kontchou and Gschwind, 1994; Radosevich et al., 1995). On the other hand, no assimilation of the ring carbon has been detected because the ring carbon atoms are at the oxidation level of CO2 (Radosevich et al., 1995), but growth using triazine-ring nitrogen has been reported (Cook and Hiitter, 1981; Mandelbaum et al., 1993a; Radosevich et al., 1995). It seems that s-triazine penetration into microbial cells is necessary before degradation (Cook, 1987). A general biodegradation pathway has been proposed for the ring of s-triazines, with cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine) as the final triazine intermediate which is then submitted to hydrolytic ring cleavage to CO2 and NH; via hydrolysis of biuret and urea, and this pathway applies to the degradation of triazine herbicides (Cook et al., 1985; Cook, 1987). Ring cleavage apparently occurs only after hydroxylation (Kaufman and Keamey, 1970). Most of the identified enzymes seem to be synthesized constitutively. Genes encoding some of these enzymes have already been cloned (Eaton and Kams, 1991). Pseudomonas sp. strain ADP mineralized atrazine through the intermediate of hydroxyatrazine (Mandelbaum et al., 1995). A DNA region,

1341

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E. Barriuso and S. Houot

encoding a putative atrazine chlorohydrolase, was recently cloned from Pseudomonas sp. strain ADP into Escherichia coli and shown to be essential for atrazine hydrolysis (de Souza et al., 1995). In the work presented here, we followed atrazine mineralization during laboratory incubations with soil samples from field experiments in Grignon, France. The soil was similar in all the experimental plots but the agricultural practices, particularly the crop rotations, were different. The [14C]C02 was measured to follow the [ring-‘4C]atrazine mineralization and related to the frequency of field atrazine application. Preliminary observations are reported on rapid atrazine mineralization in soils sampled from the field without any enrichment treatment. Complementary informations on the observed different rates of atrazine mineralization and their stability are reported. Ring mineralization was compared in other s-triazine herbicides, simazine (6chloro-N2,p-diethyl1,3,5-triazine-2,4-diamine) and terbutryn (N*-tert-butyl-$-ethyl-6-methylthio-1,3,5triazine-2,4-diamine). MATERIALS

M80 and WM73) and the bare fallow plot (B91) had received atrazine, at a rate of 1.0-1.5 kg ha-‘. The soil was sampled to a depth of 20 cm in all the plots, on different dates. All the plots were sampled in April 1994, before atrazine application. Additionally, plot B91 was sampled in July and November 1994. After sampling, the soils were immediately passed through a 5 mm sieve. Soil water contents were measured at 105°C before use. Chemicals

Analytical standards of pesticides and their metabolites were purchased from ChemService (West Chester, PA, U.S.A.). The [U-ring-‘4C]atrazine (specific activity 659 MBq mmol-‘; radiopurity of 97%) was purchased from Amersham (Buckinghamshire, U.K.). The [U-ring-i4C]simazine and [U-ring-‘4C]terbutryn (specific activities 200 and 239 MBq mmol-‘, respectively; radiopurities higher than 95%) were gifts from Ciba-Geigy (Basel, Switzerland). Solutions of [i4C]atrazine, [‘4C]simazine and [14C]terbutryn were prepared in water at 5.7, 2.4 and 5.2 mg l-t, and 3.82, 4.78 and 7.58 MBq I’, respectively. The structures of chemicals are presented in Fig. 1.

AND METHODS

Soils and experimental plots Incubation experiments

Soil samples came from the experimental farm of the National Institute of Agronomy (Grignon, France). The plots are located on a plateau. The soils, developed from a quaternary deposition of wind-blown silt, are similar and classified as Typic Eutrochrept (Soil Taxonomy) or Calcic Cambisol (F.A.O. soil classification). Six plots were selected because of their different cultivation histories and frequencies of atrazine application. Four plots were cultivated under maize: plot M62 (continuous maize since 1962), plot M80 (continuous maize since 1980), plot B91 (continuous maize between 1980 and 1991, then maintained bare fallow since 1991) and plot WM73 (annual wheat-maize rotation since 1973). Two plots were never sown with maize: plot W65 (continuous wheat since 1965) and PG (permanent grass). The main soil characteristics are in Table 1. Only the maize-cultivated plots (M62, Table Plot

Treatment

Clay

1,Main

soil characteristics Silt

Incubations were done with fresh soil samples equivalent to 40 g of dry soil, in hermetically stoppered jars at 28 + 1°C. The incubations with the soils sampled in April, July and November lasted 64, 78 and 87 days, respectively. The soil-water content was adjusted to 90% of the field capacity with 3 ml of the [14C]atrazine solution plus water. The concentration of atrazine was 0.42mg atrazine kg-’ soil which represented 0.28 MBq kg-’ soil. The incubations were run in triplicate. The evolved [i4C]C02 was trapped in 5 ml of 0.5 M NaOH, periodically sampled and replaced. Total C-CO2 was measured by calorimetry, on a continuous flow analyser (Skalar, the Netherlands) and [14C]C02 was determined, after luminescence extinction, with a scintillation counter Betamatic V (Kontron Ins.) using Picofluor (Packard) as scintillation liquid. in the different

Sand

experimental C

plots N

(B kg-‘)

PH

CaCOS

water

k kg-‘)

M62

Maize since 1962

253

623

91

Il.5

1.20

8.2

33

M80

Maize since 1980

214

718

63

10.9

1.23

7.8

6

B91

Maize/bare since 1991

171

729

86

10.8

1.20

8.0

14

WM73

Wheat-maize rotation

261

584

140

13.7

1.40

8.0

16

W65

Wheat since 1965

241

445

171

16.8

I .66

8.0

140

Permanent fx*ss

235

674

89

18.6

1.77

6.7

2

PG

Rapid ring mineralization of atrazine a~~~W”s

Y

NH’%,

Sirrrmlne

ay+-qNH~5 NHCH(CH&

AtIdlE

cH~sy?-qNH”H’ NHCH(CH&

Terbntryn

Fig. 1. Chemical structures of simazine, atrazine and terbutryn. For the April samples, in which low mineralization rates of the atrazine-ring were observed after

64 days of incubation, the incubated soils were inoculated with 5 g of soil that rapidly mineralized atrazine. The soil was then re-incubated under the same conditions for 82 additional days, and evolved [‘4C]C02 was periodically measured. The effect of soil conservation or sterilization on atrazine mineralization was studied with soil samples from plot B91. The soils sampled in April and July were kept at 4°C for 9 months. An aliquot of the soil sampled in April was air-dried and stored for 4 months at room temperature. Gamma irradiation (30 kGy for 21 h) was used to sterilize another aliquot of the soil sampled in April. After the different treatments, the soils were then incubated with atrazine under the same conditions as previous. Incubation experiments under similar conditions were also done with [U-ring-‘4C]terbutryn and simazine and soil sampled in July in plot B91. The applied amounts were 0.18 and 0.39 mg kg-’ soil, for simazine and terbutryn, respectively. Anaiysis of [f4C]atrazine residues

Separate incubations were done under similar conditions for 41 days with 5 g of soil from plots PG and B91 sampled in April. At intervals up to 41 d of incubation, the extractable and non-extractable radioactivity was determined. Each sample was extracted with 15 ml of methanol for 24 h at 20°C three successive times. The non-extractable radioactivity, corresponding to the soil-bound residues, was determined by trapping the [14C]C02 produced after combustion with a Packard Sample Oxidizer 307 (Packard Instruments Co, Downers Grove, IL, U.S.A.) followed by liquid scintillation counting. Methanol extracts were pooled and their radioactivity determined. Then, 40 ml of the methanol extract was concentrated until dryness by evaporation under vacuum at 40°C with a Rotavapor (Btichi RE 111). Residues were dissolved in 2 ml of methanol and [‘4C]atrazine and 14C-labeled metabolites were analyzed by HPLC on a Novapak Cl8 column (Waters, 5 pm, 250 mm x 4.6 mm), with a Waters instrument (600E System controller, and 717 radioactive flow detector Autosampler) and (Packard-Radiomatic Flo-one A550). The mobile phase was methanol-water buffered with 50 mM ammonium acetate with pH adjusted to 7.4. The

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chromatography started with 40/60 methanol-water (v/v) for 14min then methanol-water (65/35) for 30 min. The mobile phase flow was 1.0 ml min-‘, and the injected sample volume varied between 100 and 300 ul depending on the radioactivity content of each sample. RESULTS AND DISCUSSION

Mineralization of the triazine-ring of atrazine in the different soils

The kinetics of atrazine mineralization in soils sampled in April in the different plots are presented in Fig. 2. Three different types of behaviour were observed. In plots M62, MS0 and B91, 75% of the initially applied atrazine was mineralized at 64 days. In these plots, the mineralization rate increased rapidly at the beginning of the incubations, reaching a plateau after 43 days. In plots W65 and PG, less than 4% of the applied atrazine was mineralized. In plot WM73, intermediate amounts of atrazine mineralization were observed; the mineralization increase at the beginning of the incubation was less important than in plots M62, M80 and B91, but after 64 days of incubations the kinetics had not reached a plateau and [‘4C]COz continued to be evolved. Atrazine mineralization rates were related to the soil cropping history. Indeed, the highest rates of atrazine mineralization occurred in plots under continuous maize with annual atrazine application. No differences were found in soils from plots receiving atrazine since 1962 or 1980 (respectively M62 and M80, B91). The least atrazine mineralization occurred in plots never cultivated with maize, which consequently never received atrazine. In plot WM73 under a wheat-maize rotation and receiving atrazine every two years, intermediate amounts of atrazine mineralization were observed. This plot was cultivated with maize the year before the sampling date. Atrazine mineralization showed a lag phase of several days at the beginning of the incubations with the soils characterized by rapid atrazine mineralization (M62, M80 and B91). The mineralization

0

e

I

h,ti?

Fig. 2. Kinetics and rates of atrazine-ring mineralization during laboratory incubations (28”C, soil moisture quivalent to 90% of the field capacity) in soil sampled in April in the diffetent experimental plots. The confidence intervals were smaller than the symbol size.

1344

E. Barriuso and S. Houot

rate reached a maximum between 8 and 15 days of incubation, corresponding to a mineralization of 44.5% of the applied atrazine d-‘. The mineralization rate then decreased, corresponding to the plateau observed in the mineralization kinetics. Such shapes of mineralization kinetics could indicate a microflora adapted to the mineralization of the atrazinering. The initial lag phase would correspond to the growth of the microbial population. In plot WM73, the maximum rate of mineralization was retarded between 15 and 22 days and remained lower than 2% of the applied atrazine d-‘. After 28 days, the mineralization rate was higher in WM73 than in M62, M80 and B91. As after 64 days of incubation, the total mineralization only reached 50% of the applied atrazine but was still increasing, the microbial community involved could have been different than previously. The low and rather constant mineralization rates observed in plots W65 and PG indicated the absence of a specific microflora adapted to atrazine-ring mineralization. More work about the active microorganisms would be necessary to clearly explain these differences. These results could be explained by the development of a microflora able to degrade atrazine, and could be related to the repeated atrazine applications to the soils. There is an extensive literature on the enhanced microbial degradation of pesticides in soil following repeated application of the same pesticide. Concerning herbicides, these phenomena are mainly reported with phenoxyalkanoic acids and carbamates (Roeth, 1986), and with some others compounds as linuron, propyzamide, metamitrone, and mainly, napropamide (Walker and Welch, 1992). However, to our knowledge, this is the first report of enhanced atrazine degradation in soil after repeated field applications. Rapid mineralization of the triazine-ring of atrazine has been notified in recent works after microbial enrichment during laboratory experiments (Gschwind, 1992; Mandelbaum et al,, 1993a, 1995; Radosevich et al., 1995). The different rates of atrazine mineralization could hardly be related to differences in the physico-chemical characteristics of the soils, Particle size distributions were roughly similar in all the plots. Soil pH in plots with high atrazine mineralization rate was around 8; only plot PG. under permanent grass, had a pH lower than 7. However atrazine mineralization seemed independent of the soil pH as low rates were also observed in plot W65 for which pH was 8. The atrazine mineralization decreased when soil organic C and N increased. It has been demonstrated that atrazine sorption increased with soil organic matter content (Barriuso and Calvet, 1992) and atrazine bioavailability for microbial degradation decreased when sorption and bound residue formation increased (Barriuso et al., 1994).

Fig. 3. Kinetics and rates of soil organic carbon mineralization during laboratory incubations (28”C, soil moisture equivalent to 90% of the field capacity) in soil sampled in April in the different experimental plots. The confidence intervals were smaller than the symbol size.

The kinetics of total soil organic carbon mineralization (Fig. 3) reflected the global microbial activity. No latency time was found in the overall respiration, and the respiration rate was maximum after 1 day of incubation in all the soils. These kinetics were not related with those of the atrazine mineralization due to a specific biological activity. Mineralization zincs

of the s-triazine-ring

in different tria-

Mineralization of the s-triazine-ring in other triazines was measured during incubations of the B91 soil with ring-14C labeled terbutryn (a thiomethyl-striazine) and simazine (a chloro-s-triazine) (Fig. 4). The ring mineralization kinetics for simazine were similar to those of atrazine. Mineralization was rapid at the beginning and then reached a plateau corresponding to 61% of the applied simazine. In contrast, after 68 days of incubation of the same soil with terbutryn, the ring mineralization represented only 14% of the initial amount. These results indicated that in this soil, triazine ring mineralization would probably be dependent upon the chloro-substitution, as with a thiomethyl group instead of chlorine, rapid mineralization was not observed. It has been shown recently that dechlorination was the first step of atrazine degradation in soils and was of microbial origin (Mandelbaum et al., 1993b; de Souza et al., 1995) when it was

Fig. 4. Mineralization kinetics of the triazine-ring in atra-

zine, simazine and terbutryn during laboratory incubations in soil from the B91 plot sampled in July. The confidence intervals were smaller than the symbol size.

Rapid ring mineralization of atrazine widely accepted to be a soil-catalyzed chemical process (Skipper et al., 1967). Ring cleavage apparently occurs only after hydroxylation (Kaufman and Kearney, 1970), and rapid mineralization of triazine-ring was often observed with triazine hydroxyderivatives (Cook, 1987). Extractable atrazine residues were analyzed during incubations with soils B91 and PG, which showed rapid and slow atrazine mineralization rates respectively (Fig. 5). Throughout the two incubations, the extractable radioactivity remained mostly as atrazine and few quantities of metabolites were detected. The main metabolites were dealkylated derivatives and the amount of hydroxyatrazine was very low. In the incubation with the PG soil, less than 2% of the initial radioactivity was mineralized, 40% was methanol extractable and 59% were stabilized as bound residues in the soil. Atrazine represented 56% of the extractable residues corresponding to 23% of the initial applied amount. The main metabolite was the deethylatrazine. Its pro-

0

10

20

1345

portion remained constant after 7 days of incuother metabolites bation. The appeared progressively during incubation. After 41 days of incubation, the metabolites represented, in % of the applied atrazine: 1.l for hydroxyatrazine, 4.7 for deethylatrazine, 3.5 for deisopropylatrazine and 4.7 for deethyldeisopropylatrazine. In the incubation with the B91 soil, 69% of the initial radioactivity was mineralized. Less radioactivity remained extractable and stabilized as bound residues than in the PG soil (respectively 13 and 19% of the initial radioactivity). Again, atrazine was the main constituent of the methanol extract and represented 69% of the extractable radioactivity at the end of the incubation corresponding to 9% of the applied atrazine. The more important metabolite was the deethylatrazine during the Iirst 10 days of the incubation, but decreased progressively during the incubation while the proportion of the deethyldeisopropylatrazine progressively increased and became the most important metabolite. At the

30

40

‘rime WYS) Fig. 5. Fate of [ring-14C]atrazine in laboratory incubations in soils from plots B91 and FG sampled in April.

1346

E. Barr&o and S. Houot

end of the incubation, the metabolites represented, in % of the applied atrazine: < 0.1 for hydroxyatrazine, 0.2 for deethylatrazine, 0.2 for deisopropylatrazine and 2.3 for deethyldeisopropylatrazine. From the results presented in Fig. 5, two competitive dissipation processes could be proposed for the atrazine in the studied soils: rapid dissipation through ring cleavage and mineralization in the plots receiving atrazine every year, and more progressive dissipation through bound residues formation in the other soils. No evidence of the atrazine pathway of degradation could be deduced from our results. Little hydroxyatrazine was detected. Thus, if hydroxylation was a necessary step for ring cleavage and mineralization, it probably occurred inside the microbial cells with only liberation of [‘4C]C02, the last step of atrazine mineralization. Otherwise, hydroxylation could be a limiting step in atrazine degradation; as hydroxylated atrazine would be degraded very rapidly. Topp et al. (1995) did not find any accumulation of hydroxyatrazine or dealkylated atrazine in sediments with high capacity for atrazine mineralization and concluded similarly. On the contrary, Maldelbaum et al. (1993a,b) and Assaf and Turco (1994) observed the transient accumulation of hydroxyatrazine. of‘ Variation sampling date

atrazine

mineralization

with

Fig. 7. Kinetics of atrazine-ring mineralization in W65 and PG soil samples inoculated with soil from plot B9l and incubated under laboratory conditions. All samples were incubated with atrazine for 64 days before inoculation. In the inoculated samples, the [‘4C]C02 evolved from the B91 inocuhnn was subtracted. The confidence intervals are

shown when larger than the symbol size.

the

The bare plot B91 was sampled in April, before field atrazine application, in July, 2 months after atrazine application, and in November. The three corresponding kinetics of atrazine mineralization during laboratory incubations are presented in Fig. 6. In July, the mineralization rate reached 82% of the initial radioactivity, with a very short latency time at the beginning of the incubation. Atrazine mineralization in the April sample presented a longer lag phase with a final mineralization rate of only 74% after 64 days of incubation when it was 81% after the same time of incubation in the July soil sample. In the November soil sample, the mineralization was very slow at the beginning of the in-

Fig. 6. Kinetics and rates of atrazine-ring mineralization in soils from plot B91 sampled in April, July and November. The confidence intervals were smaller than the symbol size.

cubation and the mineralization plateau was not reached after 87 days of incubation. A seasonal variation in the responsible microbial activity could be possible but these preliminary observations need further studies. Possible tivity

inoculation

of atrazine

mineralization

ac-

Experiments were conducted to study the potential inoculation of atrazine mineralization in soil. In April, after 64 days of incubation with atrazine, samples from the PG and W65 plots were inoculated with soil from plot B91, also incubated with atrazine, and atrazine-ring mineralization measured during 82 more days of incubation. In Fig. 7, the increase of atrazine mineralization in plots PG and W65 was calculated after correction of the [‘4C]COz coming from the B91 soil inoculum, considering that the rate of atrazine mineralization was similar in the B91 sample incubated alone at the same time. The atrazine mineralization increased in the two soils, and it exceeded 15% of the initially applied atrazine. The increase of mineralization was less pronunced than at the beginning of the incubation with the B91 sample (Fig. 2). Two complementary reasons could be proposed: the microbial population responsible for atrazine mineralization had a limited possibility to colonize another soil, or the atrazine availability decreased during the first two

Rapid ring mineralization of atrazine

Fig. 8. Effect of y-irradiation on atrazine-ring mineralization in B91 soil incubated in sterile conditions or inoculated with a fresh B91 soil suspension in water. The kinetics of atrazine mineralization in the fresh B91 soil used for inoculation are given as reference. The confidence intervals are shown when larger than the symbol size.

months of incubation before inoculation (Barriuso et al., 1992). After soil sterilization by gamma irradiation, the atrazine-ring mineralization disappeared in the B91 soil (Fig. 8). It indicated that the enzymes involved in atrazine-ring mineralization were not exocellular as gamma irradiation does not denaturate the enzymes present in the bulk soil and that mineralization needed a biological activity. The inoculation with fresh B91 soil suspension in water partially restored the atrazine-ring mineralization but the mineralization rate remained lower than in the corresponding soil used for inoculation. It could denote the low competitivity for substrate colonization of the microorganisms responsible for atrazine mineralization. Modification

of the potential

atrazine

mineralization

during soil storage

Atrazine mineralization strongly decreased in the April sample after soil storage during 9 months at 4”C, but not in the July sample (Fig. 9). The soil water content was only 8% of soil weight in the July sample, and 18% in the April sample. Soil sto-

1347

rage at 4”C, under moist conditions modified more strongly the active microorganisms than under dry conditions. The microorganisms adapted to cold temperature could better develop in humid conditions and would become the dominant and active microorganisms. Air-drying in laboratory conditions until a residual moisture of 2% provoked a loss of atrazine mineralization capacity in the B91 soil sampled in April (Fig. 9). The corresponding mineralization kinetics was nearly linear and the total atrazine-ring mineralized after 63 days of incubation was only 12% of the applied atrazine, when 14% of the initial atrazine were mineralized in the fresh soil sample. In conclusion, the atrazine-ring mineralization capacity seemed related to the field atrazine application frequency. Rapid atrazine-ring mineralization was observed in soils without previous enrichment. The mineralization of the triazine-ring was related to the presence of a chloro-substituent: rapid mineralization was also observed with simazine, another chloro-s-triazine, but not with terbutryn, a thiomethyl-s-triazine. In the studied soils, two competitive dissipation processes were proposed: rapid dissipation through ring cleavage and mineralization in the plots receiving atrazine every year, and more progressive dissipation through bound residues formation which seems to be the general behaviour in most soils. The atrazine-ring mineralization was of microbial origin. This microbial activity varied during the year and was rather sensitive to soil storage in cold and humid conditions. The responsible microorganisms were not competitive and their activity very sensitive to changes in soil conditions. More work has already be undertaken to characterize the responsible microorganisms and their activity. Acknowledgements-Thanks

are due to Jean-Noel Rampon for technical assistance, Jacques Troizier for field management, and Dr Edward Topp for reading the manuscript.

REFERENCES

Fig. 9. Kinetics of atrazine-ring mineralization in fresh soils sampled in the B91 plot in April and July, then in the same soils after 9 months of storage at 4°C and in the April sample after 4 months of dry storage. The confidence intervals are shown when larger than the symbol size.

Assaf A. A. and Turco R. F. (1994) Accelerated biodegradation of atrazine by a microbial consortium is possible in culture and soil. Biodegradation 5, 29-35. Barriuso E. and Calvet R. (1992) Soil type and herbicides adsorption. International Journal of Environmental Analytical Chemistry 46, 117-128. Barr&o E., Koskinen W. and Sorenson B. (1992) Modification of atrazine desorption during field incubation experiments. The Science of the Total Environment 123/W, 333-344. Barriuso E., Benoit P. and Bergheaud V. (1994) Role of soil fractions in retention and stabilisation of pesticides in soils. In Environmental Behaviour of Pesticides and Regulatory Aspects (A. Copin, G. Houins, L. Pussemier and J. F. Salembier, Eds), pp. 138-143. European Study Service, Rixensart, Belgium.

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and S. Houot and carbofuran in soil. Soil Biology and Biochemistry 25, 1097-I 105. Mandelbaum R. T., Wackett L. P. and Allan D. L. (1993a) Mineralization of the s-triazine ring of atrazine Applied and by stable bacterial mixed cultures. Environmental Microbiology 59, 1695170 1. Mandelbaum R. T., Wackett L. P. and Allan D. L. (1993b) Rapid hydrolysis of atrazine to hydroxyatrazine by soil bacteria. Environmenral Science and Technology 27, 194331946. Mandelbaum R. T., Allan D. L. and Wackett L. P. (1995) Isolation and characterization of a Pseudomonas sp. that mineralizes the s-triazine herbicide atrazine. Applied and Environmenral Microbiology 61, 1451-1457. Mouain C.. Lauaero C.. Asther M.. Dubroca J., Frasse P. and Asther-M. (1994) Biotransformation of the herbicide atrazine by the white rot fungus Phanerochaete chrysosporium. Applied and Environmenral Microbiology 60, 7055708. Radosevich M., Traina S. J., Hao Y. L. and Tuovinen 0. H. (1995) Degradation and mineralization of atrazine by a soil bacterial isolate. Applied and Environmental Microbiology 61, 297-302. Roeth F. W. (1986) Enhanced herbicide degradation in Reviews of Weed Science 2, soil with repeat application. 45-65. Skipper H. D., Gilmour C. M. and Furtick W. R. (1967) Microbial versus chemical degradation of atrazine in soils. Soil Science Society of America Proceedings 31, 653-656. Topp E., Gutzman D. W., Bourgoin B., Millette J. and Gamble D. S. (1995) Rapid mineralization of the herbicide atrazine in alluvial sediments and enrichment cultures. Environmental Toxicology and Chemistry 14, 743747. Walker A. and Welch S. J. (1992) Further studies of the enhanced biodegradation of some soil-applied herbicides. Weed Research 32, 19-27. Gschwind N. ( 1994) Yanze-Kontchou C. and Mineralization of the herbicide atrazine as a carbon source by a Pseudomonas strain. Applied and Environmental Microbiology 60, 4297-4302.