Transformacje mikrobiologiczne niektórych herbicydów fenyloamidowych w glebie

R. B AR TH A

MICROBIAL TRANSFORMATIONS AND ENVIRONMENTAL FATE OF SOME PHENYLAMIDE HERBICIDES

Department of Biochemistry and Microbiology Rutgers University, New Brunswick, N.J., USA

In the global ecosystem, one of the most essential functions of micro­ organisms is the recycling of complex organic molecules to their mineral constituents. Given favorable environmental conditions, the tremendous flexibility of microbial metabolism provides for the biodégradation of virtually all natural as well as most man-made organic compounds. The failure o f microorganisms to degrade certain organochlorine insecticides such as DDT at an acceptable rate created a novel environmental pollution problem. By now, the ecological danger of recalcitrant lipo­ philic biocides is well recognized, and has led to the restriction or bann­ ing of these compounds in the USA and in other countries.

A novel and as yet less well recognized situation arises when a "bio­ degradable” pesticide contains molecular subunits that are recalcitrant. In such cases bioassays and chemical analyses specific for the parent^ compound will indicate degradation, occasionally at a very rapid rate.’ A more thorough study may reveal, however, that a substantial portion of the pesticide molecule is still in the environment. The chemical be­ havior and toxicity characteristics of the metabolite may be quite distinct from those of the parent compound, and often radiotracer studies are needed to monitor the full range o f transformation products and their respective persistence. The environmental fate of some aniline-based herbicides serves to illustrate this point.

Phenylamide herbicides have in common an aniline ring, usually substituted in one or more positions. According to the nature of their aliphatic moiety, the phenylamides are subdivided into phenylurea, phe- nylcarbamate and acylanilide herbicides (Fig. 1). Together, these com­ pounds constitute a very substantial and economically important portion

pehnylcarbamates are readily attacked by microorganisms, and the first degradative step is the cleavage o f the N— С bond followed by the ox i­ dation of the aliphatic moiety [11, 20]. The phenylureas are degraded more slowly and a stepwise déméthylation precedes the cleavage of the N— С bond [19].

In our investigations on the fate and effects of phenylamide herbicides in soil we employed the C 0 2-evolution method [8] supplemented by gas and thin layer chromatography, mass spectrometry and other micro- analytical techniques [1, 9]. The C 0 2-evolution of treated soil as com ­ pared to untreated controls reveals whether or not the pesticide serves as a carbon source for soil microorganisms, whether it exerts any in­ hibitory effect, or exhibits some combination of these possibilities. Figure 2 shows the effect of some model substances on the C 0 2-evolution of soil. Predictably, addition of a carbon source (glucose) increases C 0 2- evolution temporarily, and a persistent inhibitor (NaN3) causes a lasting depression of C 0 2-evolution. Phenol, which is an inhibitor but also a potential carbon source, initially depreses C 0 2-evolution but as phenol degraders become selectively enriched, a large burst of C 0 2-evolution follows. 3,4-Dichloropropionanilide (propanil), a selective herbicide used principally for the control of barnyard grass (Echinochloa crus-galli) in rice (Oryza sativa), exhibited yet another C 0 2-evolution pattern (Fig. 3): an initial increase followed by persistent inhibition. This pattern was

P henylurea Phenylcarbamate Acylanilide

Monuron N 0 CH3 /= \ I II / C l - \ J - N - C - N ' CH3 Diuron H 0 CH3 1 II / * c i- Q - n- c- n^ а снз Neburon H 0 CH3 z=\ I II / C l - Ç j - N - C - N / C l \Н 9 C hlorpropham H 0 /=\ I II / J - N - C - O - C H CH3 / C l \ CH3 Swep H 0 /=\ I II C l - \ J - N - C - 0 - C H 3 /

a

Fig. 2. The effect of glucose (Gl) sodium azide (AZ) and phenol (Ph) on C 0 2 evolution by soil. The soil samples (50 mg by dry weight Nixon sandy loam) were moistened to 60% of their water holding capacity. A ll treatments were at the 500 ppm level. To accentuate the effects, C 0 2 evolution of the untreated

control was substrated from that of the treated samples (A C 02)

Fig. 3. The effect of 500 ppm S'^'-dichloropropionanilide (propanil) on the C 0 2- - evolution of soil. Soil samples and plotting as in Fig. 2

interpreted as indicating the cleavage and oxidation of the propionate moiety of propanil, while the 3,4-dichloroaniline (DCA) moiety was thought to persist and to exert an inhibitory effect [6]. Gas chromato­ graphic analysis of the residues revealed an even more complex si­ tuation: DCA was further transformed to 3,3',4,4'-tetrachloroazobenzene (TCAB) and other polymeric products (Fig. 4). The latter transformations i are unusual, since they are synthetic rather than degradative in nature. Nevertheless, the formation of these and additional polymerization pro­ ducts were independently observed also by other investigators in the laboratory as well as in the field [18, 22, 24, 26]. Similar transformations were found to affect several structurally related acylanilides and phe- nylcarbamates, but not phenyluręas [1, 3, 10]. TCAB and the other poly­ meric products are stable compounds and persist for several years in soil [18, 24].

Microbial activity was required both for the initial cleavage of pro­ panil as well as for the polymerization step. By classical enrichment techniques, it was possible to isolate the microorganisms responsible for the cleavage step [25], but isolation of the TCAB-formers was poss­ ible only after the enzymatic mechanism (peroxidation) was discerned [7] allowing the design of a specific isolation procedure [12]. With the help o f this procedure, peroxidase-producing microorganisms in soil were enumerated. Their numbers and the peroxidase activity measured directly on soil showed positive correlation with the capacity of the soil sample to form TCAB from DCA [5, 14].

lecular features that render an aniline compound subject to enzymic, transformation to azobenzenes and other polymeric residues. For this: reason we systematically tested the transformation of variously

sub-C l - Q - N H - sub-C 0 - sub-C H 2- ch3 /

a

CI_ / = \ _ w =N - { 4 - N - 0 - C T + other Polyaromatic

\ Products

a

Cl

ą - f34— Oichloroanilino ) - 3,3' 4 ' - Trichloroazobenzene

Fig. 4. Simplified pathway of the microbial transformations of 3',4'-dichloropropion-anilide (propanil) in soil

stituted anilines in soil (Fig. 5), and later in a reaction mixtjure con­ taining the purified peroxidase and oxidase o f an isolated soil fungus

Geotrichum candidum [15]. The results of these studies showed that

susceptibility to enzymic transformation was positively correlated with increasing electron density at the amino group. Anilines substituted by electron-withdrawing groups in both ortho (2,6- or non-adjacent ortho and meta (2,5-) positions resist transformation [16].

The transformation of anilines to azobenzenes involves labile inter­ mediates such as the free anilino radicals and the phenylhydroxylamine analogs [13, 17]. Since these labile intermediates react randomly with unchanged aniline residues, multiple herbicide applications will result in hybrid azobenzene residues [2], as illustrated on Fig. 6.

At low herbicide concentrations the chemical binding o f aniline re­ sidues to the soil organic matter effectively competes with the poly­ merization process [4, 18]. Experiments with 14C-ring-labeled

chloro-Fig. 5. Formation of azobenzenes from anilines by microorganisms in soil as influenced by the type, number and position of substituents. The benzene ring with question mark indicates the formation of unidentified polyaromatic products. ’’None” sands for no detectable transformation in 14 days. Identification of products was based on gas and thin layer chromatography using appropriate standards.

Note the partial dechlorination of 2,3-dichloroaniline

and to purified humic and fulvic acid preparations. Fractionation of the soil organic matter-chloroaniline complex indicates that at least two types of bonds exist in these complexes: hydrolyzable (probably amide) and non-hydrolyzable (probably ether) bonds [21].

c i- { j - n h- co- c2h5 / « C l Propan/I CH3 h3c- Q - n h- co- c h- c3h7 / n _Solan

a - s j - N = N - s j - a

ci

Fig. 6. Pathway of ’’hybrid residue” formation from S'^'-dichloropropionanilide (propanil) and N-(3-chloro-4-methylphenyl)-2-methyl-pentanamide (solan). Both herbicides are licensed for use on the same crop and thus their residues have

opportunity to react with each other

Polymerization of chloroanilines to azobenzenes and the chemical binding of chloroanilines to humic compounds both act to greatly retard the mineralization (conversion to C 0 2) of herbicide-derived chloroaniline residues. Projected measurements of 14C 0 2-release indicate that the complete elimination of aniline residues from a single application at recommended levels may require up to 10 years. With repeated herbicide applications, a build up of polymerized and humus bound aniline residues in agricultural soils may occur. Since the continued and perhaps ex­ panded use of phenylamide herbicides is to be expected, it appears advisable to stay alert for the possibility that they may contaminate agricultural produce or influence soil fertility.

REFERENCES

[1] B a r t h a R. J.: Agr. Food Chem. 16, 1968, 602-604. [2] B a r t h a R.: Science 166, 1969, 1299-1300.

[3] B a r t h a R.: Weed Sei. 17, 1969, 471, 478.

[5] B a r t h a R., B o r d e l e a u L. M.: Soil Biol. Biochem. 1, 1969, 139, 143. [6] B a r t h a R., L a n z i l o t t a R. P., P r a m e r D.: Appl. Microbiol. 15, 1967, 67-75. [7] B a r t h a R., L i n k e H. A. В., P r a m e r D.: Science 161, 1968, 582-583. [8] B a r t h a R., P r a m e r D.: Soil Sei. 100, 1965, 68-70. [9] B a r t h a R., P r a m e r D.: Science 156, 1967, 1617-1618.

[10] B a r t h a R., P r a m e r D.: Bull. Env. Contam. Toxicol. 4, 1969, 240-245. [11] B a r t h a R., P r a m e r D.: Adv. Appl. Microbiol. 13, 1970, 274-275.

[12] B o r d e l e a u L. M., B a r t h a R.: Appl. Microbiol. 18, 1969, 317-341. [13] B o r d e l e a u L. M., B a r t h a R.: Bull. Env. Contam. Toxicol. 5, 1970, 34-37. [14] B o r d e l e a u L. M., B a r t h a R.: Can. J. Microbiol. 18, 1972, 1857-1864. [15] B o r d e l e a u L. M., B a r t h a R.: Can. J. Microbiol. 18, 1972, 1865-1871. [16] B o r d e l e a u L. M., B a r t h a R.: Can. J. Microbiol. 18, 1972, 1873-1882. [17] B o r d e l e a u L. M., R o s e n J. D., B a r t h a R.: J. Agr. Food Chem. 20,

1972, 573-578.

[18] С h i s а к a H., K e a r n e y P. C.: J. Agr. Food Chem. 18, 1970, 854-858. [19] G e i s s b ü h l e r H.: In ’’Degradation of herbicides” , P. C. Kearney and

D. D. Kaufman (eds.), Dekker, New York 1969, 79-111.

[20] H e r r e t t R. A.: In ’’Degradation of Herbicides”, P. C. Kearney and D. D. Kaufman (eds.), 1969 New York, 113-145.

[21] H s u T., B a r t h a R.: Soil Sei. 1973, in press.

[22] K e a r n e y P. C., P l i m m e r J.: J. Agr. Food Chem. 20, 1972, 584-585. [23] K e a r n e y P. C., P l i m m e r J. R., G u a r d i a F. S.: J. Agr. Food Chem.

17, 1969, 1418-1419.

[24] K e a r n e y P. C., S m i t h R. J., Jr, P l i m m e r J. R., G u a r d i a F. S.: Weed Sei. 18, 464-466, 1970.

[25] L a n z i l o t t a R. P., P r a m e r D.: Appl. Microbiol. 19, 1970, 301-306. [26] P l i m m e r J. R., K e a r n e y P. С., C h i s a к a H., Y o u n t J. В., К 1 i n-

g e b i e l U. I.: J. Agr. Food Chem. 18, 1970, 859-861.

*

Paper of the Journal Series, New Jersey Agricultural Experiment Station New Brunswick, N.J. The described investigations w ere supported by the U.S.P.H.S. research grant ES-16 and by the P.R.P. NE-53. Pre­ paration and delivery of this paper was aided by a grant of the Rutgers University Research Council.

R. B A R T H A

TRANSFORMACJE M IKROBIOLOGICZNE

NIEKTÓRYCH HERBICYDÓW FE NYLO AM ID O W YCH W GLEBIE

Katedra Mikrobiologii i Biochemii, Uniwersytet Rutgersa, New Brunswick, USA

S t r e s z c z e n i e

Mikroorganizmy glebowe mogą rozrywać wiązanie N— С niektórych herbicy­ dów acylamilidowych i fenylkarbaminowych. Grupa alifatyczna zostaje utleniona,

benzenów i ich polimerów lub zostaje chemicznie wiązana z połączeniami humi- nowymi. Obydwa te procesy powodują dużą trwałość pozostałości herbicydu w środowisku. Ze względu na powszechne stosowanie tych herbicydów możliwość nagromadzania się pochodnych anilinowych powinna być brana pod uwagę i ściśle kontrolowana. Р. Б А Р Т А МИКРОБИОЛОГИЧЕСКАЯ ТРАНСФОРМАЦИЯ ФЕКИЛАМИДОВЫ Х ГЕРБИЦИДОВ В ПОЧВЕ Кафедра микробиологии и биохимии, Радгерс университет, Ню Джерси, США Р е з ю м е Почвенные организмы могут разрывать соединения N— С ацсланилидовых и фенилкарбаминовых гербицидов. Амифатическая группа окисляется, но отделённый анилин трансформируется энзиматическим путём до азобензена и его полимеров или-же химически соединяется с гуминовыми соединениями. Оба эти процесса являются причиной большой прочности остатков гербицидов в почве. Принимая зто влияние а также широкое применение выше указанных гербицидов следует считаться с возможностью их накопления в почве.