Pestic. Sci. 1982, 13, 253-256

Transformation of the Herbicides Propanil and Chlorpropham by Micro-algae

S. John L. Wright and Andrew Maule

School of Biological Sciences, University of Bath, Bath, Avon BA2 7A Y

(Revised manuscript received 3 June 1981)

Two species of green algae and four of blue-green algae hydrolysed the acylanilide herbicide propanil to the aniline derivative, 3,4-dichloroaniline. Of the cultures tested, only the blue-green alga Anacystis nidulans was shown to be capable of con- verting the phenylcarbamate herbicides propham and chlorpropham to the corres- ponding anilines. The green alga UIothrixJimbriata was apparently unable to hydrolyse propanil or chlorpropham.

1. Introduction

A common feature of the microbial transformation of herbicides that contain a phenylcarbamoyl group is their conversion to the corresponding aniline compound.1,2 Although a body of informa- tion now exists3 concerning transformations of such herbicides by bacteria and fungi, the algae have been largely ignored. This is surprising in view of the physiological affinities of algae with plants. Furthermore, compounds such as chlorpropham and propanil are used for weed control in paddy

Following the observation that some blue-green algae, particularly non-axenic cultures, could transform the acylanilide herbicide propanil to 3,4-di~hIoroaniline,~ a wider range of axenic algal cultures have been examined for their ability to transform this and other compounds containing a phenylcarbamoyl group. In these studies, herbicide-transforming activity was determined as the release of the corresponding aniline compound.

where conditions are conducive to algal growth.

2. Experimental methods 2.1. Algae Axenic cultures of the green algae Chlorella pyrenoidosa 21 1/8h, Ulothrix fimbriata 38412 and Chlamydomonas reinhardii 1 1 /32a, and the blue-green algae Gloeocapsa alpicola 143011, Anacystis nidulans 14051 1, Anabaena cylindrica 1403/2a, Tolypothrix tenuis 148213a and Nostoc muscorum 1453112 were obtained from the Culture Centre of Algae and Protozoa, Cambridge. They were maintained by monthly transfer on slants of the appropriate media (see later) solidified with pure agar (15 g litre-I), and incubated at room temperature with daylight illumination.

2.2. Culture methods Green algae were grown in a Knops ~o lu t ion ,~ G. alpicola, An. nidulans and A . cylindrica in the modified medium of Hughes et al.,s and T. tenuis and N . muscorum in medium D plus H5 micro- elements solution. For growth under nitrogen-fixing conditions the nitrates in the media*eQ were replaced by a corresponding amount of sodium sulphate.

Batch cultures (100 ml) were grown in Erlenmeyer flasks (250 ml) from 5-ml inocula sampled in the exponential growth phase. Suspensions of those algae which tended to clump (T. tenuis and N. muscorum) were homogenised by stirring prior to use as inocula. Green algae were incubated

0031-613X/82/06o(M253 $02.00 0 1982 Society of Chemical Industry

253

254 S. J. L. Wright and A. Made

at 25°C under constant illumination (warm-white fluorescent, 4000 lx) on an orbital shaker (110 rev min-1) and blue-greens at 30°C with constant illumination (warm-white fluorescent, 1400 lx) and shaken at 100 rev min-l. Confirmation that algal cultures remained axenic throughout the study was obtained by frequent microscopic checks and by using nutrient agar as a test medium.

2.3. Herbicides Propham (99% purity) was obtained from the Sigma London Chemical Co. Ltd, Poole, England. Purified propanil and chlorpropham (99.9% purity) were respectively donated by Rohm & Haas, Philadelphia, USA, and Mirfield Chemical Co. (now Ciba-Geigy Agrochemicals, Cambridge, England). Solutions of the herbicides were prepared in the algal growth media and sterilised by membrane filtration by the methods previously described.63'0 Sterile herbicide stock solutions were stored in the dark at room temperature.

2.4. Herbicidealgal suspension incubation systems In initial tests for herbicidetransforming activity, dense algal suspensions were prepared by pooling cells harvested from two 100-ml 10-day cultures and resuspending them in 100 ml of fresh medium. The suspension (5 ml) was added to 5 ml of herbicide solution (50 pg ml-I), giving 25 pg ml-1 in the reaction mixture, and incubated in 50-ml Erlenmeyer flasks at 30°C (blue-green algae) or 25°C (green algae) in the light or dark (wrapped in aluminium foil) on an orbital shaker (60 rev min-1). After 24 h the flask contents were centrifuged (60009 for 15 min) and the supernatant phase assayed for the presence of aniline compounds. Cultures giving a positive result in the foregoing procedure were examined further. Cell suspensions harvested from four 100-ml cultures were resuspended in 150 ml of fresh medium to give between 0.7 and 0.9 mg dry weight of cells ml-l. Samples (10 ml) of the algal suspension were incubated for up to 96 h with herbicide incorporated (25 pg ml-1) as before. Controls comprised algae or herbicides incubated alone.

2.5. Detection of aniline compounds Aniline determinations were based on diazotisation and coupling to produce an azo dye. 3,4- Dichloroaniline (3,4-DCA) in the supernatant phases of algae-propanil reaction mixtures was determinedll using 1-naphthol as the coupling agent and measuring the absorbance of the pink colour at 510 nm. Aniline and 3-chloroaniline (3-CA) in the supernatant phases of reaction mixtures containing propham and chlorpropham were determined12 using N-1-naphthylethylenediamine dihydrochloride and measuring the purple colours at 555 and 540 nm, respectively. Although these colorimetric methods are not specific, it is reasonable to assume in the circumstances that the appropriate aniline compound was being determined. The limit of detection of the anilines was of the order of 0.05-0.1 pg ml-l.

2.6. Thin-layer chromatography Dense suspensions were prepared by resuspending the cells from 200 ml of culture in 15 ml of medium. The suspensions contained 10.5 mg dry weight of cells ml-l for An. nidulans and 4.2 mg mi-' for C. i-einhardii. Portions ( 5 ml) of the suspension were incubated with herbicide solution (5 ml; 50 pg ml-1) for 3 days and the supernatant phases collected after centrifugation (6000g for 15 min); these were analysed by thin-layer chromatography (t.1.c.) using silica gel PF254 (Merck) plates (0.3 mm thickness), activated at 100°C for 30 min. For propanil and 3,4-DCA, the extractions and subsequent t.1.c. in one direction with benzene+acetone (95+5 by volume) were as described in the method of Lanzilotta and Pramer.13 Propanil and 3,4-DCA spots were located under ultraviolet light (350 nm). Chlorpropham and 3-CA were extracted and chromatographed using the method of Clark and Wright.14 Plates were developed in one direction using petroleum spirit (distillation range 40-60"C)t- di-isopropyl ether (1 + 1 by volume); the spots were located under ultraviolet light (350 nm).

Transformation of herbicides by micro-algae 255

3. Results

All the algae tested, except U.j?mbriata, formed 3,4-DCA from propanil in the initial tests (Table l), but only An. nidulans produced detectable amounts of 3-CA from chlorpropham. Only blue-green algae were tested with propham; again An. nidulans was the sole alga to form aniline. No aniline compound was detected in the control flasks.

3-CA was rapidly produced from chlorpropham by An. nidulans incubated in the dark and production was maintained at a higher level than that produced in illuminated cultures (Figure 1). The amount of 3,4-DCA formed from propanil by T. tenuis was of the same order as that of 3-CA from chlorpropham by An. nidulans, and was similar in the dark and the light (Figure 2). The activity was, however, significantly lower than that of C. reinhardii.

An extract of the An. niduluns-chlorpropham incubation mixture gave two spots by t.1.c. One of these, RF 0.43, corresponded with 3-CA (RF 0.38) and the other, RF 0.82, with chlorpropham

Table 1. Transformation of propanil and chlorpropham to 3,4-dichloro- aniline and 3-chloroaniline by algal cell suspensions

Alga

Cloeocapsa alpicola Anacystis nidulans Anabaena cylindrica Anabaena cylindricab Tolypothrix tenuis Tolypothrix renuis Chlamydomonas reinhardii Ulothrix fimbriata Chlorella pyrenoidosa

Transformation4

Propanil to 3,4-dichloroaniline

Chlorpropham to 3-chloroaniline

(6 + =metabolite detected; - =metabolite not detected. * Culture grown under nitrogen-fixing conditions.

r 2

8 . 0 1

c 6 .0 -

4.0-

2 . 0 -

8 .0

6 .0

4.0

2.0

0 20 40 60 A0

/

Time ( h ) Time ( h )

Figure 1. Transformation of chlorpropham to 3-chloroaniline by Anacystis nidulans. Algal cell suspensions (0.7 mg dry weight ml-1) were incubated in duplicate at 30°C with chlorpropham (25 pg ml-1): A , in the light; and 0, in the dark.

Figure 2. Transformation of propanil to 3,4-dichloroaniline by Chlamydomonas reinhardii and Tolypothrix tenuis. Suspensions of C. reinhardii cells (0.9 mg dry weight ml-1) were incubated in duplicate at 25°C with propanil (25 pg ml-1): A, in the light; and 0 , in the dark. T. renuis cells (0.77 mg dry weight ml-1) were incubated at 30°C with the same concentration of propanil: A, in the light; and 0, in the dark.

256 S. J. L. Wright and A. Maule

(RF 0.82). No such spots were obtained from the incubation of An. nidulans alone, whilst an extract of a solution of chlorpropham, incubated without algae, gave one spot, RF 0.81, corresponding with chlorpropham. Analyses of the C. reinhardii-propanil extract by t.1.c. supported the high level of propanil-hydrolysing activity (Figure 2), in that no propanil was detected. Only one spot, RF 0.47, was detected, corresponding with 3,4-DCA (RF 0.48). Extracts from C. reinhardii, incubated without propanil, gave no spots, whilst incubation of propanil without algae gave one spot, RF 0.26, corresponding with the propanil standard ( R F 0.26). These results indicated that 3-CA and 3,4-DCA were formed by these algae in the transformation of chlorpropham and propanil, respectively.

4. Discussion This survey has indicated that some green algae and blue-green algae (cyanobacteria) can trans- form propanil and chlorpropham by releasing the corresponding aniline compound. This trans- formation route is also common to bacteria and fungi.

It seems likely that propanil, which was particularily susceptible, was hydrolysed by acylamidase activity, as was found with the fungus Fusarium solani.I3 An acylamidase could also be responsible for the hydrolysis of c h l o r p r ~ p h a m . ~ ~ The detected aniline compounds are less phytotoxic14 and less algicida16p16 than the parent herbicides. Both propanil and chlorpropham have been shown to be inhibitory to the growth of cultures of the algal species used in this study. It is proposed to report on these phenomena in a separate paper. It should be stated, however, that the inhibitory effects were noted using culture conditions differing from those reported herein, primarily with lower cell densities.

The literature is noticeably short of reports of herbicide degradation studies using algae. Only one such study with a compound containing a phenylcarbamoyl group is known to the authors. In this," it was found that the green alga Chlorella fusca produced several metabolites from the substituted urea compound [14C]buturon, although these did not include an aniline derivative. This raises the possibility that the algae used in the present study may have produced metabolites other than anilines. However, the specific objective of this study was to determine whether algae, like other microbes, are able to form anilines from herbicidcs that contain a phenylcarbamoyl group.

Acknowledgement Part of this work was undertaken whilst A. Maule was in receipt of a research studentship from the Natural Environment Research Council.

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