ARAUJO, W. L.

LUCHINI, L. C.

MELO, I. S. de

PROCOPIO, A. R. de L.

PROCOPIO, A.R. de L.

MELO, I.S. de

LUCHINI, L.C.

ARAUJO, W.L.

RCAAP - Repositório Científico de Acesso Aberto de Portugal

Mineralization of the herbicide C-Propanil in soil by Pseudomonas putida AF7.

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, Proceedings of lhe 1dh Intematíonsl Conference on Environmenlal Science and Techno/ogy Kos island, Greece, 5 - 7 September 2007 MINERALlZATION OF THE HERBICIDE 14C_PROPANIL IN SOIL BY Pseudomonas putida AF7 A.R. PROCÓPI01; 1.5. MEL01; L.C. LUCHINI2 and W.L. ARAUJO' 'Laboratory of Environmental Microbiology, Embrapa, CP. 69, CEPo 13820-000 Jaguariúna, SP, Brazil; 2 Biological Institute, São Paulo, SP; 30epartment of Genetics, ESALQ, São Paulo University, Piracicaba, SP, Brazi!. e-mail: Itamar@cnpma.embrapa.br AB5TRACT Many pesticides are persistent in soil environmental and the persistence depends on the dissipation processes. The herbicide propanil has been used extensively for the selective control of broad-Ieaved weeds in rice fields. Its widespread use has caused environmental concern on the basis of frequent detection in surface water and groundwater. In this wOrk, it is described the isolation of rhizobacteria from rice roots and the investigation of the mineralization of 14C-propanil in soi!. Extensive biodegradation of propanil by a strain of Pseudomonas putida AF7 was demonstrated in nutrient cu~ures. The organism was capable of using propanil as the sole source of C, presenting up to 60% of degradation. Ring cleavage was confirmed in 14C02 evolution experiments. Between 17 and 19% of ring - 14C was mineralized to 14C02 in natural soil and in soil inoculated with the bacteria, respectively. The formation of extractable metabolites of propanil were detected in natural soil (18%) and in soil supplemented with P. putida this value was of 20%. On the oIher band, bound residues were formed in higher amount in natural soil, while in soil with P. putida was a little smaller. These resu~s suggest that it may be possible to develop practical systems based on the use of this bacteria to detoxify propanil-contaminated soi!. Keywords: Biodegradation, Propanil, Pseudomonas, Mineralization, Bound Residues. 1.INTRODUCTION Propanil (3', 4' - dicloropropionanilide) is a herbicide extensively used around the world for control of weed in rice productions. Propanil and its metabolites are transported within characteristic ditch ecosystems in the production landscape of southeast Brazi!. In soil, biodegradation of propanil liberates 3,4-dicloroaniline, which is converted by microbial peroxidades to 3'3,4,4'-tetrachloroazobenzene (TCAB) and oIher azo products. The TCAB may accumulate in rice-growing soils, and leach into groundwater. Studies have shown continued degradation into TCAB and TCAOB. There is great concem regarding TCAB and TCAOB because of embriolethal, teratogenic, and possible genotoxic effects shown in toxicity tests [1]. Repeated applications of herbicides are sometimes required during crop rotation and these may promole an increase in soil microorganisms able to at!ack a particular herbicide, a process known as enhanced degradation. Few studies on propanil degradation, its transport in soils and plants have been performed in tropical soils. The objectives of this study were to: i) quantify 14C labeled propanil mineralization, and; ii) determine bound and extractable residues in soil samples A-1218 2. MATERIAL ANO METHOOS 2.1 Mineralization of 14C Propamil The biodegradation of the herbicide propanil was evaluated in soi!, supplemented with 14C-propani! diluted in technical grade propanil and inoculated with a strain of Pseudomonas putida AF7. This strain was isolated from the rhizosphere of rice grown in contamination soil with this herbicide, in southeast of Brazil. Technical grade 14C-ring labeled propanil, tested and confirmed to have a radiochemical purity of 98% and specific activity of 32.4 KBq.mg was obtained from Sigma Chemical Company. The supplementation solution was prepared using both labeled and únlabeled chemical. Fifty grams (dry weight) of soil (four replicates) was placed into biometric f1asks [2J. 14C_ propanil was added at final concentration of 7.43 KBq and 0.525mg.mL". Vials containing solution of 0.2 KOH were placed in each f1ask to trap CO2 evolved during the incubation period. The solution ofKOH was collected after 1, 3, 5, 7,14,21 and 28 days. 2.2 Extractable and Bound Residues Soluble residues were extracted from dried milled soi! (50g) using methanol. The supernatants of each sample were combined and the radioactivity was measured in a liquid scintillation counter. The initial radioactivity and remaining after the extracted residues was determined by combustion of 500 mg (dry weight) soil samples in a Biological Oxidizer - OX 600 and the total C02 evolved was trapped in a monoethanolamine solution. 3. RESUL TS ANO OISCUSSION In laboratory tests P. putida AF7 was able to degrade over 50% of propanil when evaluated by HPLC. At the same time, this bacteria produced high amount of indole acetic acid. When inoculated In seeds of rice, the strain promoted lhe plant growth and colonized the root system. The process of colonization of root was monitoring using a field emission scanning electron microscopy (Zeis + Leica) (Figure 1). The "C02 data presented enable comparison of the rates of propanil mineralization in non-sterilized soi!. Mineralization of propanil was high within 14 days. After 14 days ofthe incubation the mineralization was reduced (Figure 2), suggesting an accelerated degradation in the early period. The 14C02 production from 14C-propanil supplemented into the soil was 18.67% in natural soil infested with the bacteria and 17.02% in soil without the bacteria (Figure 3). Figure 1. Colonization of rice roots by Pseudomonas putida AF7, viewed by a field emission electron microscopy. The images show the bacteria colonizing the root hair of rice. A-1219 a) General of the root tip, where the bacteria was observed b) Colonization of Pseudomonas cells in the rool hair. Image shows the attachmenl of the bacleria and biofilm formation. ~ ;g • -'2 8 .. '" 5 e'" u 4 :\ ""' l-o-· P+B+E I .. P+E I ___ ,_ I o 2 t' '" .. o o '"' ~ T1 T3 T5 T7 T14 T21 T28 Time (days) Figure 2. Evolution of 14C from 14C-propanil in soil inoculated with Pseudomonas putida. '§ '" o ... "" • U :\ ""' ot' '" 6 '"' ~ 20% 15% 10% 5% 0% T1 T3 T5 T7 T14 T21 T28 Time (days) t+-P';Ei:"E'., • P+E I ---,--Figure 3. Evolution of 14CO, accumulaled from 14C-propanil in soil inoculated with Pseudomonas putida. The values obtained from 14C exlractable and bound residues are presented in Figures 4 and 5. In 24 hours after incubalion was possible observe the produclion of 14CO, (0.035%) and, immedialely after 48 hours, lhe produclion of CO, presented an increase of about 10 times, suggesting the start of an accelerated degradation. The produclion of 14CO, accumulaled in soil as bound residues was of 2.77% after 28 days of incubalion. Thus, the biodisponibility of residues was of 8.34%, fraclion of lhe molecule that may be degraded by lhe microorganisms. A-1220 14COZ Accumulated 30 'C <: - -~. ~ 2.5 = o ~. O> u ----i ,Q 2.0 ..... ~ ... :l ...-/./ .... O> ~ 1.5 - I .... '" ~./ .... ... = -----J ~ ::: 1.0 O> ~ -.--..------ I ... '" 0.5 ~-~ = . .--- '=J 0.0 -~-T1 T3 T5 T7 T14 T21 T28 Time (days) Figure 4. Evolution of 14CO, of bound residues using Pseudomonas putida AF7. T1 T3 T5 T7 Time (days) I I --~-.~I ------1 T14 T21 T28 Figure 5. Evolution of 14CO, from 14C of bound residues in soil inoculated with Pseudomonas putida. 4. CONCLUSION The mineralization of propanil was higher in soil inoculated with Pseudomonas putida than in soil without the baeteria. The bound residues of this herbicide were of approximately 2%. This work shows the potential of Pseudomonas putida for bioremediation of contaminated soils with pesticides. This bacterial strain also promotes the plant growth. REFERENCES 1. POTHULURI, J.; HINSON, J. A; CERNIGLlA, C E. (1991). Propanil: loxicological characterislics, metabolism, and biodegradalion polenlial in soil. J. Environ. Qual. 20:330-347. 2. BARTHA, R.; PRAMER, D. (1965). Fealures of a ftask and melhod for measuring lhe persistence and biological effecls of peslicides in sai I. 50il 5cience. v.1 00, n.1, p .68·70. A·1221 

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Mineralization of the herbicide C-Propanil in soil by Pseudomonas putida AF7.

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