A model for the formation and degradation of bound residues of the herbicide 14C-isoproturon in soil

~)

Chemosphere,Vol.

39, No. 4, pp. 627-639, 1999 © 1999 Elsevier Science Ltd. All rights reserved 0045-6535/99/$ - see front matter

Pergamon

PII: S0045-6535(99)00128-9

A MODEL FOR THE FORMATION AND DEGRADATION OF BOUND RESIDUES OF THE HERBICIDE 14C-ISOPROTURON IN SOIL

S. Reuter 1'2, M. Ilim3, J.C. Munch 1, F. Andreux2 and I. Scheunert I

~GSF - Institute of Soil Ecology, D-85764 Neuherberg, Germany. 2Universit6 de Bourgogne, Centre des Sciences de la Terre, Geo-Sol, 6 bd Gabriel, F-21000 Dijon, France. 3Turkish Atomic Energy Authority, Ankara Nuclear Research and Training Center, 06983 Saray, Ankara, Turkey.

Dedicated to Prof. F. Korte on the occasion of his 75th birthday

Abstract The humic monomer catechol was reacted with 14C-isoproturon and some of its metabolites, including 14C-4-isopropylaniline, in aqueous solution under a stream of oxygen. Only in the case of 14C-4-isopropylaniline, incorporation in oligomers, in fulvic acid-like polymers, and in humic acid-like polymers was observed. The main oligomer was identified by mass spectrometry as 4,5-bis-(4isopropylphenylamino)-3,5-cyclohexadiene-l,2-dione. Oligomers and polymers containing bound 14C-4-isopropylaniline were subjected to biodegradation studies in a loamy agricultural soil during 55 days by quantifying 14CO2 evolved. In all cases, significant mineralization rates could be determined, which, however, were much smaller than those of free 14C-isoproturon and free ~4C-4-isopropylaniline in the same soil. © 1999 Elsevier Science Ltd. All fights reserved Key words: isoproturon, phenylurea herbicide, 4-isopropylaniline, biodegradation, bound residues, humic substances, catechol, quinone.

Introduction Soil-bound residues of pesticides in general are those residues that are not extractable by organic solvents and, therefore, are not detected in normal residue analysis. They represent a soil burden, whose quantitative extent and importance is not known. In order to assess their potential ecotoxicological and toxicological

627

628

risks, the bioavallability to soil microorganisms and biodegradation must be studied. This implies the knowledge of binding mechanisms and chemical structures of bound products. The phenylurea herbicide isoproturon has been reported to form considerable amounts of unextractable residues in soils, whose levels vary in dependence of agricultural management conditions [1] and humification of organic matter [2]. In a model system with soil samples, 14C-residues, formed after the application of 14C-ring-labelled isoproturon, were incorporated twice as much in the humic acid fraction than in the fulvic acid fraction; however, it was not possible to identify the binding mechanisms [3]. The chemical identity of soil-bound residues was studied in case of other phenylurea herbicides such as monolinuron and buturon [4]; after extraction with supercritical methanol, soil-bound residues were shown to contain chloroaniline metabolites. Although these chloroanilines were detected as free metabolites in cultures of microorganisms [5], they were detected in soil in a free form only in traces [6] or not at all [7]. For isoproturon, the metabolite 4-isopropylaniline was detected in a model soil system only in traces [8]. It is assumed that aniline metabolites, after their formation in soil, are immediately bound in an unextractable form and thus not detectable as free compounds. In some studies reported in the literature, the incorporation of chlorinated anilines in soil constituents was investigated with various humic monomers, using enzymatic [9,10] or abiotic catalysts [11,12] occurring in soil. The formation of oligomers and polymers containing the xenobiotic by covalent bonds was observed. Based on these results, also for isoproturon it may be concluded that at least part of its soil-bound residues consist of the metabolite 4-isopropylaniline covalently bound to humic acid building blocks. In this paper, catechol was chosen as humic substance precursor. The binding of 14C-isoproturon, of its two demethylated metabolites, and of its metabolite tac-4-isopropylaniline to catechol and their incorporation into the humic-like polymers formed is studied. The stability of these polymers in soil and their bioavailability to microorganisms are investigated in biomineralization experiments.

Materials and Methods Chemicals

Catechol (Merck-Schuchardt, Germany, purity > 99%) was recrystallized in n-hexane. Isoproturon (IPU) [N-(4-isopropylphenyl)-N',N'-dimethylurea] (Dr. Ehmnstorfer, Germany; purity > 99%), t4C-ring-labelled isoproturon (Institute of Isotopes of Hungarian Academy of Science, Hungaria; purity > 98%), 4-isopropylaniline (4-IPA) (Merck-Schuchardt, Germany; purity >99%), 14C-ring-labelled 4-isopropylaniline (Sigma Chemical Company, USA; purity > 98%), monodemethyl-isoproturon (MD-IPU) [N-(4-isopropyl)-phenyl)-N'-methylurea], and didemethyl-isoproturon (DD-IPU) [N-(4-isopropylphenyl)urea] (Dr. Ehrenstorfer, Germany; purity > 99%) were used as purchased.

629 Synthesis conditions and product extraction

The general conditions for the synthesis of model humic substances and product extraction are based on Adrian et al. [11] and Vrlkel et al. [12] but were modified as follows: 0.025 mM 14C-IPU and 0.05 mM

MD-IPU, DD-IPU and 14C-4-IPA, respectively, were dissolved in 1.5 ml ethanol and added to 150 ml of 0.5 M phosphate buffer solution (bidistilled water, pH 7.0) containing the previously dissolved eatechol (1.5 or 3 mM) to obtain a ratio of 1 : 60 for the parent chemicals. The reaction occurred under a stream of oxygen with constant stirring at room temperature and in the dark. After 120 h the reaction mixture was filtered under vacuum through a filter funnel with a fritted glass (Goosch filter, porosity 5), and the filter funnel was rinsed successively with 0.1 N NaOH, bidistilled water, dichloromethane, and acetone. The dichloromethane phase was treated with anhydrous sodium sulfate. For the following mass spectrometry (MS) analysis the dichloromethane was evaporated to dryness under a stream of nitrogen and the obtained residues were redissolved in acetonitrile. The water phase was acidified to pH 1.5 with 5 N HCI to precipitate the acid-insoluble fraction (synthetic humic acids, HA). After centrifugation, the acid-soluble supernatant (synthetic fulvic acids, FA) was purified by dialysis (2000 Da) with distilled water. The precipitate was redissolved in NaOH (pH 8) and likewise dialysed (3500 Da). The dialysis water and the dialysed HA- and FA-solutions were repeatedly extracted by shaking with dichloromethane (4:1 / v:v) to determine unreacted or weakly bound 14C-IPU and metabolites. The organic extracts were treated with anhydrous sodium sulfate, evaporated to dryness with a rotary evaporator, and the residues were redissolved with methanol for the following high pressure liquid chromatography (HPLC) analysis. Recovery of MD-IPU and DD-IPU was 92.1 and 93.1%, respectively. Radioactive test chemicals were quantified by liquid scintillation counting (Tri-Carb 1900, Packard, Canberra). Therefore, 1 ml of liquid sample was mixed with 15 ml scintillation cocktail (Ultima Gold XR, Packard). A synthesis without pesticide molecules was performed to obtain reference samples for analytical application. After extraction of the HA- and FA-solutions with dichloromethane, their volume was reduced to 50 ml in a rotary evaporator. Kinetic experiments allowed to study the incorporation of the test chemicals in the different product fractions. The chemicals were added at progressive steps of the polymerisation reaction of catechol (0, 3, 6, 18, 24, and 48 h after the starting-point). The yield of FA and HA was determined by drying an aliquot at 105°C.

Analytical methods

Mass spectrometry was performed by ionspray ionisation with direct sample injection on a TripelQuadrupol-MS API 300 LC/MS/MS-system, PE SCIEX (Ionisation voltage 4800 V, Orifice Voltage 50 V, focus ring 400 V and -400 V for positive and negative mode, respectively).

630 HPLC was performed with a model L-7100 pump with L-7200 autosampler (Merck Hitachi) connected to a LiChrospher 100 RP-18 column (Merck). Detection was achieved with a UV/VIS-detector L-7400 (Merck Hitachi) at 240 nm and a radio detector LB 507B (Berthold). Isoproturon and its metabolites were eluted with bidistilled water/acetonitrile (gradient grade, Merck) in a gradient program and identified according to Schuelein et al. [13].

Biodegradation studies

The soil samples for the biodegradation studies were taken from one homogeneous soil sample that was collected in August from the surface horizon (0-20 cm) of a loamy agricultural soil in Lorraine, eastern France, and sieved to 2 mm without previous air-drying. Its main characteristics were: pH (CaCI2) 6.1, 1.40% Corg, 0.16% Ntot, 18% clay, 57% silt, 25% sand, 29% WHC, water content 43% of WHC. The biodegradation studies were performed in a closed laboratory system which was discontinuously aerated with humid air as described by Lehr et al. [14]. The incubation took 55 days at 22°C in the dark. Each incubation unit (100 ml) contained a 50 g soil sample (dry matter equivalent). The 14CO2 originating from the biodegradation of the 14C-labelled test chemical was trapped in 15 ml 0.1 N NaOH; then, an aliquot of 3 ml was mixed with 15 ml scintillation cocktail (Ultima Flo FA, Packard) to determine the radioactivity by liquid scintillation counting. The tested chemicals were 14C-IPU, ~4C-4-IPA and tac-humic-like polymers. The latter were chosen according to the results of the kinetic studies (table 2). Their specific radioactivity and their application rate to the soil are listed in table 3. The application rate of the lac-labelled IPU and 4-IPA was 0.022 lamol g-t soil (dissolved in acetone), equivalent to 4.52 ~g g-1 (specific radioactivity 381 Bq p.g-l) and 2.96 ~tg gq (specific radioactivity 1879 Bq p.g-l), respectively. The chemicals were first added to one sixth of the 50 g soil sample. The solvents were evaporated and the complete 50 g soil sample was homogenized and adjusted to 60% of WHC. Because of the different solvents used for the applications, each soil sample was treated with all the solvents in the same manner (3 ml acetone, dichloromethane, and water, including the solvent containing the test chemical) to avoid changing side-effects due to the solvents. At the end of the biodegradation studies, soil samples were extracted by over-head shaking (6 h) once with a 0.01 M CaC12-solution (1:1.25 / w:v) followed by methanol (1:0.8 / w:v) in six consecutive repetitions. The residual radioactivity in the soil samples (bound residues) was determined by combustion (Oxidizer 306, Packard), trapping the 14CO2 in Carbo-Sorb (Packard), and mixing with Permafluor E+ (Packard) prior to liquid scintillation counting.

631 Results

Characterization of addition product The synthesis of hurnic-like polymers in the presence of 14C-IPU, MD-IPU, and DD-IPU did not result in an important disappearance of the tested chemicals. These were recovered exclusively in the dichloromethane extracts of the dialysis water at a rate of 99.4%, 95.7%, and 93.2%, respectively. Conversely no free 14C-4-IPA could be detected in any of the extracts, but an orange coloured product carrying about 2/3 of the initial radioactivity was retained on the fritted glass filter, eluted with dichloromethane and analysed by ionspray mass spectrometry (table 1). The ionpeaks m/z = 375 and m/z = 373 for positive and negative mode, respectively, were detected, but no peak was related to 4-IPA or catechol. After mass fragmentation the spectra showed the loss of -CO and -CO-CHO fragments from the quinoid structure [12], of one or more -CH3 groups from the isopropyl-group, and of a whole phenylisopropyl-(-CgHll and -C9Hl2)-grou p. Additionally, it was observed that the hybrid product lost at least one phenylisopropylamino-(-C9Hl2N and C9Hl3N)-group. The latter was also observed as a single peak, corresponding to 4-IPA.

Table 1: Ionspray fragmentation mass spectra of the addition product of catechol and 4-isopropylaniline. positive mode m/z

composition

fragment

375 347

C24H26N202+H + C23H26N20+H +

[M+H] ÷ [M+H-CO] +

332 303 213

C22H23N20+H + C21H22N2+H + CI4H14NO+H +

[M+H-CO-CH3] + [M+H-CO-CHO-CH3] + [M+H-CO-C9HI2N] +

or C13HI2N20+H +

[M+H-CO-CH3-C9-H 1l]+

negative mode Im/z 373 358 345 330 253 239

composition

fragment

C24H26N202-H+ C23H23N202-H+ C23H26NzO-H+ C22H23N20-H+ C 15Hj4N202-H+ C15HI4NO2-H+

[M-H] [M-H-CH3] [M-H-CO]" [M-H-CO-CH3]" [M-H-C9H12]" [M-H-C9H12N]

or

210

CI4HI2N202-H +

[M-H-CH3"C9H11]"

C 13HILN2O-H+

[M-H-CO-CH3-C9HI2 ]"

OF

134

Cl4 HI3NO-H+ CgH13N-H+

[M-H-CO-C9HI3N ]" [M-H-C15HI3NO2]"

632 The hybrid molecule was identified as a trimer, the disubstituted ortho-quinone

4, 5-bis-(4-isopropylphenylamino)-3, 5-cyclohexadiene-l, 2-dione as shown in figure 1.

0

L H3C

CH3

Figure 1: Proposed structure for the trimeric addition product of catechol and 4-isopropylaniline, based on mass spectra.

Influence of retarded 14C-4-IPA addition on product formation The retarded addition of 14C-4-IPA to the reaction medium greatly influenced its incorporation in different product fractions (table 2). The results indicate that the incorporation of 14C-4-IPA, that was added at the initial step of the polymerization reaction (t = 0 h), was preferably in the trimer. The later the 14C-4-IPA was added, the smaller was the incorporation in the trimer; the incorporation reached a minimum when 14C-4-IPA was added at t = 18 h. On the contrary, the incorporation of 14C-4-IPA in the HA reached its maximum after addition at t = 24 and 48 h. Finally, the incorporation in the FA did not show a definite trend in relation to the time the 14C-4-IPA was added. The ~4C-humic-like polymers used in the biodegradation studies were synthesized according to the kinetic studies t = 0 h and t = 24 h to yield polymers with a maximum of 14C-4-IPA incorporated. Their yield and rate of 14C-4-IPA incorporation are shown in table 3.

633 Table 2: Incorporation rate of 14C-4-isopropylaniline (14C-4-IPA) ill the fractions of the catechol-derived polymers in relation to the time of 14C-4-IPA addition to the reaction medium. Values in % of the initial radioactivity. FA: model fulvic acids; HA: model humic acids; NCF: non-eharacterised fractions (in acetone, in dialysis water, and in dichloromethane extracts of FA- and HA-solutions). Time t [h]

0

3

6

18

24

48

Tdmer FA HA NCF

65.8 1.7 14.6 11.4

48.3 1.3 21.9 28.2

13.7 1.2 35.2 42.8

<0.1 2.0 59.1 37.5

<0.1 2.2 65.8 28.3

<0.1 1.0 62.8 29.6

Recovery

93.5

99.7

92.9

98.6

96.3

93.4

Table 3: Characteristics of the addition products of catechol and 14C-4-isopropylaniline (14C-4-IPA). FA: model fulvic acids; HA: model humic acids. Time of 14C-4-IPA addition and product

product yielda) [mg]

product specific radioactivity [Bq lag-l]

t4C-4.IPA rate of product incorporated in the application to soil [~g g'~] product [lag rag"]

t = 0 h b) Trimer FA HA t=

5.7 28.5 100.1

1081 4.2 17.9

720 2.8 12.0

4.1 d)

22.8 118.7

12.3 45.5

7.3 27.1

78.40 109.4 c0

24 h c)

FA HA

a)corresponding to 150 ml reaction medium b)initial specific radioactivity: 1497 Bq ~tg"l 4-IPA C)initial specific radioactivity: 1679 Bq p.g-t 4-IPA d)equivalent to 2.96 ilg 4-IPA g-i or 0.022 ~tmol4-IPA gq e)equivalent to 0.57 ~tg 4-IPA g-i or 0.004 p.mol4-IPA gd

Biodegradation studies The production of 14CO2 from the 14C-ring-labelled test chemicals during 55 days decreased significantly in the order: free IPU, free 4-IPA, 4-IPA bound in humic-like polymers (table 4). The cumulative 14CO2 production curves showed a sigmoid, a sublinear, and a linear shape, respectively (figure 2). The distribution of the extractable and non-extractable t4C-residues in the soil samples is presenteA in table 4. In all experiments more 14C-residues could be extracted with methanol, whereas the water extractable 14C-residtms declined sharply in the soil samples with 14C-4-IPA and 14C-humic-like polymers as compared to I4C-IPU.

634 In contrast, the methanol extractable 14C-residues were highest after soil incubation with free 14C-4-IPA. Among the 14C-hum±c-like polymers, the trimer showed the highest yield after methanol extraction. The formation of bound 14C-residues was stronger after application of the laC-humic-like polymers.

14

[

.¢......z

"5 o~

---~--IPU

- - - o - - FA

4

z,Z

o

0

10

20

30

40

50

60

time [d]

Figure 2: Cumulative 14CO2 production of 14C-labelled free isoproturon, free

14

•

• •

C-4-1sopropylamlme,

and 14C-4-isopropylaniline covalently bound in hum±c-like polymers in soil samples during 55 days. n = 3 +SD.

Table 4: Balance-sheet of 14C radioactivity of biodegradation studies with 14C-IPU, 14C-4-IPA and 14C-hum±c-like polymers in soil during 55 days. a - e: indicating significant difference within the column; n = 3 +SD: p < 0.05. Cumulative 14CO2 *

14C

extracted

14C extracted

with water * # with methanol

non-

14C r e c o v e r y

extractable 14C-residues

14CO2

d "l at

day 55 #

IPU

13.16 +0.74 a

5.08 +0.33 a

12.72 +0.95 b

64.08 +2.25 c

95.05 +3.65

0.124 +0.006 a

4-IPA

9.00 +0.19 b

0.45 +0.04 b

.18.18 +0.75 a

66.51 +1.23 c

94.18 +1.03

0.079 +0.002 b

Trimer

2.36 +0.19 d

0.21 +0.01 c

7.47 +0.11 c

82.83 +0.40 b

92.87 +0.26

0.038 +0.009 d

FA

3.57 +0.17 c

0.40 +0.01 b

1.06 +0.02 e

88.31 +0.82 a

93.34 +0.96

0.054 +0.006 c

HA

1.52 +0.05 e

0.17 +0.01 c

3.06 +0.02 d

89.61 +1.27 a

94.36 +1.27

0.028 +0.001 d

* correlation of cumulative 14CO2 with 14C extracted with water: r = 0.8364, n = 15, p < 0.05 correlation of 14CO2 d "l at day 55 with 14C extracted with water: r = 0.8771, n = 15, p < 0.05

635 Discussion

Synthesis The oxidative polymerization of phenolic substances is regarded as a decisive process in the formation of humic substances in soil. The simulation of these reactions under laboratory conditions allows the synthesis of humie-like polymers [15]. By introducing pesticide molecules in the reaction medium, the polymers can be used as models to explain the formation of bound pesticide residues [11,16]. In this work, isoproturon and its two demethylated degradation products were not bound irreversibly to catechol or its polymerization products, since the added test chemicals could be recovered nearly entirely after dialysis. This indicated the presence of only weak bonds [17], as it was specified also for the interactions of isoproturon with soluble natural humic substances [18]. Conversely, 4-isopropylaniline formed nucleophilic bonds i n 4 - and 5position of the ortho-quinone originating from the oxidation of catechol (figure 1). In accordance with the reaction scheme for the addition of anilines to catechol [11 ], it is confirmed that the main reaction product of 4-isopropylaniline with catechol at a pH of 7 is a dianilinoquinone. Complementary to former work, the influence of retarded aniline addition to the reaction medium is demonstrated by the kinetic studies. The results show that 14C-4-isopropylaniline is incorporated in different product fractions at varying proportions. The decline of trimer formation after

14

•

..

C-4-1sopropylanlllne

addition retarded by a few hours could be the result of the relative proportion of the newly formed phenolic polymers. If the molecule is present at the start of the reaction, it is assumed that it reacts predominantly with monomeric ortho-quinones wich can bind a second aniline after enhanced oxidation by an excess of oxygen or by unsubstituted quinone. This is suggested by the fact that in the case of unsubstituted catechol, the addition reaction never stops at the step of mono-substituted ortho-quinone [ 11,12]. At a later step of the polymerization reaction, an increasing part of the ortho-quinone is incorporated in phenolic polymeric structures which have likewise reactive quinoidal structures [12]. Consequently, aniline or mono-substituted anilinoquinone can form an increasing number of linkages with the developing polymeric structure. It is established that the covalent linkage of aniline to the ortho-quinone described for the trimer is also present in the FA and HA. IR- and NMR-spectra of polymers originating from the addition of catechol and 4-chloroaniline indicated covalent binding of the aniline, and no signal indicating the presence of unbound, free 4-chloroaniline was detected [19]. If such an addition reaction takes place in soil, it can be regarded as a possible initial step in the formation of bound residues with soil organic matter. It is therefore suggested that the degradation of isoproturon to 4-isopropylaniline might be an important step governing the formation of bound residues of isoproturon. The ~4C-model-bound residues allowed us to study the biodegradation of the structural element 14

C-4-lsopropylanlhne in a soil sample, and to compare it with the free molecule as well as with the •

..

unchanged 14C-isoproturon.

636

Biodegradation studies The amount of Iaco2 released during 55 days of incubation increased significantly in the following order: lac-model bound residues; 14C-4-isopropylaniline; 14C-isoproturon (table 4). An important factor affecting the biodegradation of chemicals in soil is their bioavailability [20,21 ]. The bioavailability is regarded to be related to the extractability, in particular water solubility [22]. This is confirmed by the significant positive correlation of the

cumulative 14CO2production after 55 days and of the

J4CO2 production rate at the end of

the incubation time with the amount of water extractable 14C-residues (table 4). A more detailed answer to the behaviour of the test chemicals in soil can be obtained by comparing their mineralization kinetics. The shape of the cumulative 14CO2curve from the 14C-model bound residues is approximately linear (figure 2). In contrast, the cumulative 14CO2 curve from the free 14C-isoproturon has a sigmoid shape with a short initial lag phase, followed by high 14CO2production in the exponential phase, and ending in a plateau phase of decreasing mineralization rates. Such a shape is characteristic of the biodegradation of isoproturon in soils [14,23,24]. For 14C-4-isopropylaniline this curve has a sublinear shape that is characterised by an immediately high mineralization rate, followed by an asymptotic decline. The decline of the curve is assumed to be the result of the reduced bioavailability because of the rapid fixation of the reactive aniline to the soil organic matrix [25,26]. It could be demonstrated that already 24 h after application of 4-isopropylaniline to different soils only 13% to 20% of the initial amount remained extractable with organic solvents [27,28]. By a former research work using different substituted anilines including the 4-isopropylaniline, similar results were obtained, but did not show in detail the distinct sublinear curve during the initial days [29,30,31 ]. With progressive incubation time, the mineralization rate of lac-4-isopropylaniline became nearly constant and resembled that of the synthetically bound 14C-molecule (figure 2). The covalent linkage probably hindered the biodegradation of 14C-4-isopropylaniline, since the three laC-humic-like polymers showed only small differences in the 14CO2evolved as compared to that of the non-bound 14C-molecules.

Conclusion

It is concluded that the formation of bound residues from isoproturon in soil is largely dependent on the previous degradation of isoproturon into the metabolite 4-isopropylaniline. These bound residues are still bioavailable but their mineralization is markedly reduced as compared to that of the parent herbicide molecule. The results obtained with a synthetic model correspond to results reported for residues of isoproturon and other phenylurea herbicides and anilines bound in natural soils. Therefore, the model employed in this study is a useful tool in the research into soil-bound residues of xenobiotic organic substances.

637

Acknowledgements We thank Dr. W. VSlkel for assistance in polymer synthesis and Dr. G. L~Srinci for assistance in MS analyses. The research work was financially supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn.

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