41 Microbial transformation of pesticides in agricultural soil

D. LEVANON

Iiltroduction

Non-point source pollution of ground and surface water by pesticides has been recorded in agricultural areas in Europe and the US (Jamet and Delleu, 1993; Ritter, 1990). This phenomenon is causing concern, due to potential risks to public health and environmental qUality. This non-point source pollution is characterized by unidentified specific origins and low concentrations of pesticides found in contaminated water. Detailed, laborious and expensive monitoring programmes are required to assess the nature and severity of the pollution. Once water is polluted, its quantity and distribution make it virtually impossible to eradicate the very low concentrations (measured in ppb) of pesticide involved. Efforts must therefore be made to minimize the risk of penetration by the pollutant to levels below the root zone, as a result of leaching or transport by run-off surface water. Such efforts must include the development of 'best management practices' (BMP), to ensure the proper use of pesticides by the grower, with minimal hazard to water quality. Among farm management practices, tillage is one that may have a significant impact on the fate of pesticides in the soil (USDA, 1989).

The practice of reduced tillage in orchards and field crops was developed for a number of reasons. Less intense tillage reduces energy and labour costs and soil compaction. Crop residues left on the surface contribute to conditions that improve water penetration, while minimizing run-off and soil erosion, as a result of higher porosity and water holding capacity (Dick et aI., 1989). On the other hand, reduced tillage (also called conservation tillage) requires more intensive use of herbicides, to control weeds, and sometimes greater use of insecticides, fungicides, etc., to control pests and diseases that develop in the crop residues. The combination of improved water penetration and increased use of pesticides can lead to their leaching into ground water (Edwards et aI., 1992).

There is also evidence that, under reduced tillage, and especially no-tillage (NT), numerous macropores develop in the soil, resulting in faster movement of water to the sub-surface. This, too, creates the potential for increased leaching of pesticides; but the results of field studies on the topic are ambiguous. Some studies show increased leaching of pesticides under NT (Gish et al., 1991; Isensee et al., 1990; Kanwar, 1991), while others show no significant differences between tilled and untilled soils (Starr, 1990; Starr and Glotfelty, 1990). Laboratory studies of

D. Rosen et al. (eds): Modem Agriculture and the Environment, 491-499. © 1997 Kluwer Academic Publishers.

492 D. Levanon

undisturbed soil cores from the field have also produced contradictory results (Edwards et al., 1992; Fermanich and Daniel, 1991; Levanon et ai., 1993).

The respective roles of biotic and abiotic processes under NT and plough tillage (PT), and their impact on the leaching of pesticides, were studied by Levanon et al. (1994). Abiotic processes, such as adsorption/retention of pesticides, resulting from higher organic matter content, and a higher rate of biodegradation, resulting from bigger and more active microbial populations, were among the explanations for a lower degree of leaching in NT soil. Synergy between fungi and bacteria, which enhanced the biotransformation of pesticides in NT soil, was also demonstrated in this study.

The present article is a review, amended with recent results, of the effect of tillage on the biotransformation of pesticides in the soil. The role of tillage is discussed as a case study of how farm management practice can be applied to the manipulation of the desired microbial transformation of organic pollutants.

2. Materials and methods

Soil samples were taken, according to Levanon et al. (1993) from the upper layer of the soil (0-5 cm) of NT and PT plots, under permanent monoculture of com since 1974. The properties of the soil are presented in Table 1.

The following pesticides were studied: (1) Herbicides

• Atrazine (6-chloro-N-[ethyl]-N-[isopropyl]-1,3,5 triazine-2,4-diarnine); • Alachlor (2-chloro-N-[2-6-diethylphenyl]-N-[methoxymethyl] acetamide); • Metolachlor (2-chloro-N-[2-ethyl-6-methylphenyl]-N-[2-methoxy-l-

methyl-ethyl] acetamide.

(2) Insecticides

• Carbofuran (2,3-dihydro-2,2-dimethyl-7 -benzofuranul methyl carbamate); • Diazinon (O,O-diethyl-O-[6-methyl-2-{ I-methylethyl }-4-pyrirnidinyl] phosphoro­

thioate); • Malathion (diethyl mercapto-succinate s-ester O,O-dimethyl phosphorodithioate).

Table 1. Properties of the soil under NT and Pf tillage in monoculture of com since 1974.

Property

pH Organic C (%) Biomass C (J..tg g-I) Total N (%) N0I -N (mg kg-I) NH4 -N (mg kg-I) P20S (mg kg-I)

Silt loam soil: 26% sand; 57% silt; 17% clay.

Pf

6.8 1.15

420 0.112

16.2 8.1

156.1

NT

5.8 1.65

780 0.163 9.4

10.9 238.6

Microbial transformation of pesticides in agricultural soil 493

Malathion

Metolachlor

Atrazine

Carbofuran

/2 OCO.NHCH3

~O\.,CH' ~CH3

Figure 1. Molecular structure of the six pesticides. with indication of the two positions of radiolabelling with 14C (for each molecule) that were used in the present study.

Pure standard and radiolabelled pesticides were obtained from the Environmental Chemistry Lab and the Pesticide Degradation Lab, both at USDA ARS, Beltsville, MD. The molecular structures and the positions of 14C labels are shown in Figure 1. Two labelling positions were studied separately for each pesticide: position 1 -uniformly ring-labelled (for malathion-succinate label); and position 2 - side-chain label (for malathion ethyl ester label).

Selective inhibition of soil microorganisms was applied according to Levanon et al. (1994) and comprised:

(1) Control - no inhibition.

(2) Inhibition of soil fungi.

(3) Inhibition of soil bacteria.

494 D. Levanon

Microbial transformation of pesticides was studied in biometer flasks, according to Levanon et al. (1994). The transformation of each pesticide, added separately to the soil, was measured by trapping evolving 14C-C02 in a base trap, and measuring recovery by means of a scintillation counter (packard). Each pesticide was added to 50 g soil at field moisture, to a concentration of 1.0 ILg g-l, after amendment with 50 000 DPM of radiolabelled formulation (14C) and kept at 25°C for 30 days.

3. Results and discussion

The term 'mineralization' is used in this study, since all pesticides studied were uniformly ring labelled (malathion was succinate labelled), as designated by 'position l' in Figure 1. The evolution of 14C-C02 from such a formulation is an indication of the complete mineralization of the molecule. Mineralization of all the pesticides studied took place more quickly in NT than in PI soil (Figure 2). This faster mineralization can be explained by the fact that the higher content of organic matter, nitrogen and phosphorus in the upper layer of NT soil led to larger and more active populations of microorganisms, thereby creating conditions favourable to the mineralization of organic compounds, including pesticides (Levanon, 1993; Levanon et al., 1994).

The next stage of this study was to assess the relative importance of fungi and bacteria in this process. This part of the study was based on the assumption that, since populations of both fungi and bacteria are higher in NT soil, both groups probably participate in the enhanced mineralization process. This hypothesis was partly confirmed by experimental results: almost no mineralization of diazinon, alachlor, metolachlor or atrazine took place when either fungi or bacteria were inhibited (Figure 3). On the other hand, carbofuran and malathion were mineralized at the same rate as the control when fungi were inhibited, and hence by the activity of bacteria. In this series of experiments the radiolabel of the pesticide was also in position 1, so that the evolution of 14C-C02 is an indication of complete mineralization. Hence, four of the six pesticides studied were mineralized in the soil by concomitant activity of fungi and bacteria. The inability of any single strain of microorganism to mineralize xenobiotic chemicals (such as pesticides) completely, under field conditions, has been observed in many cases (Miller and Poindexter, 1994).

Some knowledge of the pathways of biotransformation of pesticides was obtained by the use of formulations radiolabelled in position 2. In this case, the evolution of 14C-C02 indicated partial mineralization of the molecule. By selective inhibition of fungi and bacteria, their role in the partial degradation (biotransformation) of each pesticide can be elucidated. It was demonstrated that, in the cases of diazinon, alachlor, metolachlor and atrazine, degradation of 'side chains' was mainly due to fungal metabolism (Figure 4). Since these pesticides were not completely mineralized when bacteria were inhibited (Figure 3), it can be deduced that the undegraded central part of the molecule, the ring, is degraded mainly by bacteria.

Microbial transformation of pesticides in agricultural soil 495

These findings allow us to formulate the probable pathway of the mineralization of these four pesticides in NT soil as follows: stage 1 - fungal metabolism of the side-chains (position 2 in Figure 1); stage 2 - bacterial degradation of the ring (position 1 in Figure 1). Results from other studies support these findings. Kaufman and Kearny (1976) found that soil fungi are able to cleave the side-chains oftriazines

Atrazine. 30 r-------------------~_,

26

~20 ~ 016 ! ~ 10

6 +---,:----- !'ia!----- /iaL::..

o ~--........ ~....,., .......

80

~60

~ ~ 40

~20

0 6

Malathion.

10 16 20 26 30 Days

Alachlor. 3.6 .,-----------------------,

3 ----- _ .. --.----.. ,- --

~2_6

~ 2 !1.6 ~ 1

0.6

o ........,~-""""-+-

Carbofuran. 36 ,----------------------,

30

~25

~ 20

! 15 ~ 10

~

6

o

50

40

~30 0

~20 ~

10

0

Diazinon.

5

Metolachlor. 2.6 .,---------------------=-,

2

~ ~ 1.6 o ! 1

0.5

Figure 2. Mineralisation of the pesticides in the soil, measured as percentage recovery of 14C of the initial amount added to NT (~) or PT (_) soil.

496 D. I..evanon

via N-dealkylation, and more recently Masaphy et al. (1993) demonstrated the same activity by white-rot fungi. However, further research is required to characterise the bacterial-fungal consortium, to identify its constituent species and their respective roles in the transformation process. Such data will contribute to the understanding of the biotransformation of pesticides in the soil, allowing prediction and eventually manipUlation in different soil environments.

Atrazine. Carbofuran.

30 40 i!' ~3O ~20 > 0 ~20 ~ 10

;I!. ; 10

0 0

Days Days

Malathion. Diazinon.

80 60

~60 i!' > ~ 40 040 0

~20 ~20 ;I!. ;I!.

0 0

Days Days

Alachlor. Metolachlor.

4 3 ~3 i!' > ~2 02 ~1 ~1 ;I!. ;I!.

0 0

Days Days

Figure 3. Mineralization of the pesticides in NT soil, measured as percentage recovery of the initial amount of 14C added to soil supplemented with bactericide ("3"); fungicide ("2"); control ("1 ").

Microbial transformation of pesticides in agricultural soil 497

4. Conclusion

The NT system can serve as a case study for the development of farming practices that could enhance the detoxification and mineralization of xenobiotic chemicals in the soil. The NT system in the monoculture of corn causes shifts in physical, chemical and microbiological constituents of the upper layer of the soil. The

Atrazine. ~,---------------------,

10

0~~~10~-1±6--~~~-2~5~~~~ Days

Malathion.

~

10L2~-7---1~6--~20~-a±-~30~ Days

Alachlor. 10~----------------~~

8

~ !! 6 8 ! 4 ..,.

---....-.m",m ... _._y.m__ • •... -

O~~~--~--~--~--~ 10 15 20 25 ~

Days

60

~

i 40

°30 ~ ..,.~

10

0

70

60

~60

~40 .30 ..,.

20

10

0

10

8

~

~ 6

~ 4 ..,.

0

Carbofuran.

5 10 16 20 2& 30 Days

Diazinon.

_._. __ ............ _._._._ .............. __ ._-_. -_._ ....

6 10 16 20 2& 30 Days

Metolachlor.

5 10 15 20 2& 30 Days

Figure 4. Partial mineraIization of the pesticide in NT soil, measured as percentage recovery of the initial amount of 14C added to soil supplemented with bactericide ( ); fungicide ( ); control CA.).

498 D. Levanon

conditions that develop are favourable to the adsorption and biodegradation of pesticide residues.

Hence, the conditions in NT soil resemble a biofilter for organic chemicals, in a two-stage process: adsorption and biodegradation. Other studies, under field conditions, have also demonstrated that enhanced degradation of xenobiotic organic pollutants can be achieved by the manipulation of environmental conditions, by the addition of C or N sources, minerals, air or water, etc. (Gibson and Syler, 1992). The addition of an N source and water have been used on several occasions to enhance the degradation of such contaminants as crude oil (Atlas, 1994). Hence, recent recommendations for the field evaluation of bioremedial methods have concluded that the enhancement of indigenous microorganisms should be the first option in the bioremediation of a contaminated site, and inoculation by specific degrading microorganisms should only be given second priority (Miller and Poindexter, 1994).

The prevention of pollution caused by agricultural practices should be achieved by the development of techniques that enhance the activity of the soil as a biofilter: NT management is an example by which this goal is attained, in the case of pollution by pesticide residues. Specific methods should be developed for other cases of environmental pollution resulting from farming practices. One of the most important examples, worldwide, is the use of effluents for irrigation. Such effluents contain xenobiotics that include toxic organic chemicals. Some of these, of domestic or industrial origin, are resistant to degradation in sewage treatment plants, and are therefore still found in the treated effluents used for irrigation in arid and semi-arid zones. Such chemicals, introduced by irrigation, can sometimes be found below the root zone of crops, and later reach ground water (Feigin et al., 1990).

Research is currently underway in our laboratories to develop methods to enhance the ability of the soil to act as a biofilter, and thereby to ensure minimal pollution of ground water by toxic organic chemicals that are at present to be found in domestic and industrial effluents.

Acknowledgment

I would like to thank the administration and staff of the Environmental Chemistry Laboratory, NRI, ARS, USDA in Beltsville, MD, for providing the facilities for most of the research that was conducted towards this study.

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