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Effect of The Herbicide, Benthiocarb (Thiobencarb) on Seasonal Changes in Microbial Populations in Paddy Soil and Yield of Rice Plants
EFFECT OF THE HERBICIDE, BENTHIOCARB (THIOBENCARB) ON SEASONAL CHANGES IN MICROBIAL POPULATIONS IN PADDY SOIL AND YIELD OF RICE PLANTS Sat0 K. Institute for Agricultural Research, Tohoku University, Katahira, Sendai 980, Japan
ABSTRACT Benthiocarb (S-2-chlorobenzyl diethylthiocarbamate) affected microbiological processes and growth of rice plant as follows: The herbicide enhanced the increase in numbers of heterotrophic bacteria by eight times, but sometimes in high doses caused a decline in numbers of autotrophs. Numbers of nitrifying bacteria increased after water-drainage. Large amounts (10 x recommended rate) of the herbicide stimulated the initial decrease in the amount of ammonium-N. The herbicide dissipated after water-logging and its decrease was most marked in July followed by the accumulation of dechlorinated benthiocarb in the s o i l applied with large mount of the herbicide. Yield components of rice plant differed markedly between the soil applied with a large amount of and that with the recommended rate of the herbicide.
INTRODUCTION The herbicide, benthiocarb (S-pchlorobenzyl diethylthiocarbamate) is used widely in paddy rice field in Japan. There is very few work on the effect of the herbicide on soil microorganisms compared with the work conducted on microbial degradation of the herbicide in soil (Nakamura et al., 1977; Moon et al., 1985a, 1985b). Furthermore, in some soils accumulation of a dechlorinated product of the herbicide under reductive conditions of paddy soil dwarfed rice plants (Ishikawa et al. , 1980). The present work was conducted to discover the effect of the herbicide on several bacterial groups as well as any changes in the amount of ammonium-N. In addition the dissipation of the herbicide
-335-
and production of a dechlorinated metabolite in paddy field soil was measured.
MATERIALS AND METHODS Design of experimental plots A paddy field attached to the Institute for Agricultural Research was used for field experiments. All experimental plots were suspended with barnyard manure (30 t.ha") and nitrogen-fertilizer (150 kg N.ha-') and were divided into three groups. The first group of plots was supplied with the recommended rate (r.r.) of the herbicide, the second group with 10 x the recommended rate (10 r.r.1. The third group was not supplied with herbicide. The soil of the field was alluvial and highly clay in texture. Sampling of the soil and water-drainage Soil sampling was achieved by placing a plastic cylinder over the sampling site. After removing overlying water within the enclosure the soil sample was taken by removing the whole surface of the soil to 1 cm in depth. Counting of bacterial numbers Total bacteria and Gram-negative bacteria were counted by the dilution plate method, and ammonifiers, nitrate-reducers, denitrifiers, ammonium- and nitrite-oxidizers by the most probable number (MPN) method. All cultures were incubated at 22 'C. Chemical analyses After extracting soil samples with 2.2 M KC1, ammonium-N was analyzed colorirnetrically using nitrogen autoanalyzer (TECHNICON), and nitrite- and nitrate-N also colorimetrically with Gries-Ilosvay reagent. After extraction of soil samples with acetone and hexane, benthiocarb and dechlorobenthiocarb were analyzed by a gas-chromatograph 38himadzu GC-'IA, equipped with FDP, S-filter). Estimation of yield components of rice plant After air-drying the harvested rice plant, the yield was measured.
-336-
RESULTS AND DISCUSSION Effect of benthiocarb on bacterial numbers The herbicide caused an increase in numbers of total bacteria (Fig. 1). The number o f Gram-negative bacteria changed in a similar
NI 307
t
r
_i n l1
250
0
I
2
1I
25960
BC
ABC
ABC
ABC
3
4
5
6
Fig. 1. Effect of benthiocarb on seasonal change in numbers of total bacteria (N, millions per g of dry soil). A, 150 kg N.ha-' plus 30 tons manure. ha-'; B, as A plus recommended rate (r.r.1 of benthiocarb; C, as A plus 10 r.r. of benthiocarb; 1, June I ; 2, June 19; 3, July 2 ; 4, July 24; 5, August 8; 6, September 18
-337-
fashion although the populations of Gram-negative bacteria were lower on average 10 % than that of the total bacteria. The herbicide also stimulated the initial increase in numbers of ammonifiere, nitratereducers and denitrifiers, while it depressed the initial increase in numbers of ammonium- and nitrite-oxidizers, especially in the soil with 10 r.r. of the herbicide (Fig. 2 ) .
.
306 29
I
ABC 2
1
ABC 3
ABC 4
ABC 5
1
ABC 6
Fig. 2 . Effect of benthiocarb on seasonal change in numbers of nitrite oxidizers (N, thousands per g of dry soil). Symbols as in Fig. 1. It is a well known phenomenon that the microbial population increases significantly in the soils treated with fumigants compared with not treated soil. This phenomenon has been explained as "the partial sterilization effect". In the present study the increase in numbers of heterotrophic bacteria in the soil supplied with benthiocarb has some resemblances to "the effect". In general, the herbicide stimulated populations of heterotrophic bacteria, while it inhibited those of
-338-
autotrophic ones. Therefore, it is suggested that "the effect" is dependent on the kind of bacteria. Effect of water-drainage on change in bacterial numbers in soil applied with the herbicide Water-drainage of paddy soil is sometimes conducted as practical management, to avoid dwarfing of rice plant brought about by accumulation of dechlorinated benthiocarb. In view of the management the plots with 10 r.r. of the herbicide were drained in the end of July. Waterdrainage did not affect the numbers of total and Gram-negative bacteria, although it increased slightly the numbers in September. The number of nitrite-oxidizers increased markedly in the drained plot in September (Fig. 3 ) .
I 156
50
hl52
n 1
AB 2
AB 3
AB 4
ABC 5
ABC 6
Fig. 3 . Effect od water-drainage on seasonal change in numbers of nitrifiers (N, thousands per g of dry soil). Top, nitrite-oxidizing bacteria; bottom, ammonium-oxidizing bacteria ; A , recommended rate (r.r.) of benthiocarb, flooded plot; B, 10 r.r. of benthiocarb, flooded plot; C, 10 r.r. of benthiocarb, drained plot; 1 - 6, dates as in Fig. 1.
-339-
Flooding following drainage may generate reductive condition of paddy soil, and this results in reduction of oxidative compounds produced through drainage. If the nitrifier produces nitrate during the drainage, the flooding may reduce nitrate and ultimately lead to loss of nitrogen through denitrification. Thus, one management conducted to avoid one problem may in some cases bring about another problem. Amount of ammonium-N The amounts of nitrite- and nitrate-N were very small (1.0-1.5 ppm) during the surveying period. The amount of ammonium-N decreased in all plots, and the ratio of the initial decrease was the largest in the plot with 10 r . r . of the herbicide. The decrease from August to September was greater in the drained plot than in the undrained one, suggesting nitrification by the nitrifier which increased by drainage. Benthiocarb and dechlorobenthiocarb Benthiocarb diseipated in all plots. A dechlorinated product accumulated in the plot with 10 r.r. of the herbicide corresponding to a rapid decrease of the herbicide in this plot in July (Fig. 4 ) .
Fiq. 4 . Changes in the amounts of applied benthiocarb and produced dechlorobenthiocarb in soil. H, benthiocarb (1,3) or dechlorobenthiocarb ( 2 , 4 ) in soil, ppm; 1,2 recommended rate ( r . r . 1 of benthiocarb applied; 3,4 10 r . r . of benthiocarb applied D, dates 1 - 6 as in Fig. 1.
-
-3 40-
Yield of rice plant Application of 10 r . r . of the herbicide depressed clearly the yield components. Although the yield components except ear length and ear weight were slightly smaller in the plot-B1 than in the plot-A7, the difference was not statistically significant. Excess amount of the herbicide dwarfed rice plant corresponding to accumulation of a dechlorinated benthiocarb. The dwarfing resulted seriously in the decrease in total weight, ear weight, ear length and number of grain but in number of tiller (Tab. 1). This may be interpreted as an earlier initiation of tillering rather than accumulation of the dechlorinated product. Table 1. Growth of rice plant in the respective plots of paddy field. Yield components
Plotsa A1
A7
B1
112.752 5.07b
71.332 8.45
107.80? 1.96
31.602 1.82
22.332 3.45
28.752 3.50
182.502 9.36
40.00+11.31
167.50+15.75
Ear length, cm
19.752 0.99
12.662 4.83
Ear weight, g
3.29i 0.39
0.64? 0.01
Plant height, cm Number of tillers Total weight, g
Number of grains of one ear
169.50'20.01
29.33+ 2.35
20.00:
0.89
3.49+ 1.07 155.50'15.65
a Plots B1, A1 and A7 are those of recommended rate ( r . r . 1 of, 10 times of r.r. of the herbicide, and control, respectively. Values are means standard deviation of 6 replicate samples from plots B1 and A7 and of 4 replicate samples from plot A l l respectively. This research is the part of a project, Green Energy Program (GEP-82, 83, 84-111-2-1) financed by the Ministry of Agriculture, Forestry and Fisheries of Japan.
-341-
REFERENCES ISHIKAWA, K., SHINOHARA, R., YAGI, A., SHIGEMATSU, S . , KIMURA, I.: Identification of S-benzyl N,N-diethylthiocarbamate in paddy field soil supplied with benthiocarb herbicide. J. Pesticide Sci. 5: 107-109, 1980. MOON, Y. H., KUWATSUKA, S . : Microbial aspects of dechlorhation of the herbicide benthiocarb (thiobencarb in soils. J. Pesticide Sci. 10: 513-521, 1985a. MOON, Y. H., KDWATSUKA, S . : Factors influencing microbial dechlorination of benthiocarb (thiobencarb) in the soil suspension. J. Pesticide Sci: 10: 523-528, 1985b. NAKAMURA, Y. K., ISHIKAWA, K., KUWATSUKA, S . : Degradation of benthiocarb in soils as affected by soil conditions. J. Pesticide Sci. 2 : 7-16, 1977.
ABSTRACT Benthiocarb (S-2-chlorobenzyl diethylthiocarbamate) affected microbiological processes and growth of rice plant as follows: The herbicide enhanced the increase in numbers of heterotrophic bacteria by eight times, but sometimes in high doses caused a decline in numbers of autotrophs. Numbers of nitrifying bacteria increased after water-drainage. Large amounts (10 x recommended rate) of the herbicide stimulated the initial decrease in the amount of ammonium-N. The herbicide dissipated after water-logging and its decrease was most marked in July followed by the accumulation of dechlorinated benthiocarb in the s o i l applied with large mount of the herbicide. Yield components of rice plant differed markedly between the soil applied with a large amount of and that with the recommended rate of the herbicide.
INTRODUCTION The herbicide, benthiocarb (S-pchlorobenzyl diethylthiocarbamate) is used widely in paddy rice field in Japan. There is very few work on the effect of the herbicide on soil microorganisms compared with the work conducted on microbial degradation of the herbicide in soil (Nakamura et al., 1977; Moon et al., 1985a, 1985b). Furthermore, in some soils accumulation of a dechlorinated product of the herbicide under reductive conditions of paddy soil dwarfed rice plants (Ishikawa et al. , 1980). The present work was conducted to discover the effect of the herbicide on several bacterial groups as well as any changes in the amount of ammonium-N. In addition the dissipation of the herbicide
-335-
and production of a dechlorinated metabolite in paddy field soil was measured.
MATERIALS AND METHODS Design of experimental plots A paddy field attached to the Institute for Agricultural Research was used for field experiments. All experimental plots were suspended with barnyard manure (30 t.ha") and nitrogen-fertilizer (150 kg N.ha-') and were divided into three groups. The first group of plots was supplied with the recommended rate (r.r.) of the herbicide, the second group with 10 x the recommended rate (10 r.r.1. The third group was not supplied with herbicide. The soil of the field was alluvial and highly clay in texture. Sampling of the soil and water-drainage Soil sampling was achieved by placing a plastic cylinder over the sampling site. After removing overlying water within the enclosure the soil sample was taken by removing the whole surface of the soil to 1 cm in depth. Counting of bacterial numbers Total bacteria and Gram-negative bacteria were counted by the dilution plate method, and ammonifiers, nitrate-reducers, denitrifiers, ammonium- and nitrite-oxidizers by the most probable number (MPN) method. All cultures were incubated at 22 'C. Chemical analyses After extracting soil samples with 2.2 M KC1, ammonium-N was analyzed colorirnetrically using nitrogen autoanalyzer (TECHNICON), and nitrite- and nitrate-N also colorimetrically with Gries-Ilosvay reagent. After extraction of soil samples with acetone and hexane, benthiocarb and dechlorobenthiocarb were analyzed by a gas-chromatograph 38himadzu GC-'IA, equipped with FDP, S-filter). Estimation of yield components of rice plant After air-drying the harvested rice plant, the yield was measured.
-336-
RESULTS AND DISCUSSION Effect of benthiocarb on bacterial numbers The herbicide caused an increase in numbers of total bacteria (Fig. 1). The number o f Gram-negative bacteria changed in a similar
NI 307
t
r
_i n l1
250
0
I
2
1I
25960
BC
ABC
ABC
ABC
3
4
5
6
Fig. 1. Effect of benthiocarb on seasonal change in numbers of total bacteria (N, millions per g of dry soil). A, 150 kg N.ha-' plus 30 tons manure. ha-'; B, as A plus recommended rate (r.r.1 of benthiocarb; C, as A plus 10 r.r. of benthiocarb; 1, June I ; 2, June 19; 3, July 2 ; 4, July 24; 5, August 8; 6, September 18
-337-
fashion although the populations of Gram-negative bacteria were lower on average 10 % than that of the total bacteria. The herbicide also stimulated the initial increase in numbers of ammonifiere, nitratereducers and denitrifiers, while it depressed the initial increase in numbers of ammonium- and nitrite-oxidizers, especially in the soil with 10 r.r. of the herbicide (Fig. 2 ) .
.
306 29
I
ABC 2
1
ABC 3
ABC 4
ABC 5
1
ABC 6
Fig. 2 . Effect of benthiocarb on seasonal change in numbers of nitrite oxidizers (N, thousands per g of dry soil). Symbols as in Fig. 1. It is a well known phenomenon that the microbial population increases significantly in the soils treated with fumigants compared with not treated soil. This phenomenon has been explained as "the partial sterilization effect". In the present study the increase in numbers of heterotrophic bacteria in the soil supplied with benthiocarb has some resemblances to "the effect". In general, the herbicide stimulated populations of heterotrophic bacteria, while it inhibited those of
-338-
autotrophic ones. Therefore, it is suggested that "the effect" is dependent on the kind of bacteria. Effect of water-drainage on change in bacterial numbers in soil applied with the herbicide Water-drainage of paddy soil is sometimes conducted as practical management, to avoid dwarfing of rice plant brought about by accumulation of dechlorinated benthiocarb. In view of the management the plots with 10 r.r. of the herbicide were drained in the end of July. Waterdrainage did not affect the numbers of total and Gram-negative bacteria, although it increased slightly the numbers in September. The number of nitrite-oxidizers increased markedly in the drained plot in September (Fig. 3 ) .
I 156
50
hl52
n 1
AB 2
AB 3
AB 4
ABC 5
ABC 6
Fig. 3 . Effect od water-drainage on seasonal change in numbers of nitrifiers (N, thousands per g of dry soil). Top, nitrite-oxidizing bacteria; bottom, ammonium-oxidizing bacteria ; A , recommended rate (r.r.) of benthiocarb, flooded plot; B, 10 r.r. of benthiocarb, flooded plot; C, 10 r.r. of benthiocarb, drained plot; 1 - 6, dates as in Fig. 1.
-339-
Flooding following drainage may generate reductive condition of paddy soil, and this results in reduction of oxidative compounds produced through drainage. If the nitrifier produces nitrate during the drainage, the flooding may reduce nitrate and ultimately lead to loss of nitrogen through denitrification. Thus, one management conducted to avoid one problem may in some cases bring about another problem. Amount of ammonium-N The amounts of nitrite- and nitrate-N were very small (1.0-1.5 ppm) during the surveying period. The amount of ammonium-N decreased in all plots, and the ratio of the initial decrease was the largest in the plot with 10 r . r . of the herbicide. The decrease from August to September was greater in the drained plot than in the undrained one, suggesting nitrification by the nitrifier which increased by drainage. Benthiocarb and dechlorobenthiocarb Benthiocarb diseipated in all plots. A dechlorinated product accumulated in the plot with 10 r.r. of the herbicide corresponding to a rapid decrease of the herbicide in this plot in July (Fig. 4 ) .
Fiq. 4 . Changes in the amounts of applied benthiocarb and produced dechlorobenthiocarb in soil. H, benthiocarb (1,3) or dechlorobenthiocarb ( 2 , 4 ) in soil, ppm; 1,2 recommended rate ( r . r . 1 of benthiocarb applied; 3,4 10 r . r . of benthiocarb applied D, dates 1 - 6 as in Fig. 1.
-
-3 40-
Yield of rice plant Application of 10 r . r . of the herbicide depressed clearly the yield components. Although the yield components except ear length and ear weight were slightly smaller in the plot-B1 than in the plot-A7, the difference was not statistically significant. Excess amount of the herbicide dwarfed rice plant corresponding to accumulation of a dechlorinated benthiocarb. The dwarfing resulted seriously in the decrease in total weight, ear weight, ear length and number of grain but in number of tiller (Tab. 1). This may be interpreted as an earlier initiation of tillering rather than accumulation of the dechlorinated product. Table 1. Growth of rice plant in the respective plots of paddy field. Yield components
Plotsa A1
A7
B1
112.752 5.07b
71.332 8.45
107.80? 1.96
31.602 1.82
22.332 3.45
28.752 3.50
182.502 9.36
40.00+11.31
167.50+15.75
Ear length, cm
19.752 0.99
12.662 4.83
Ear weight, g
3.29i 0.39
0.64? 0.01
Plant height, cm Number of tillers Total weight, g
Number of grains of one ear
169.50'20.01
29.33+ 2.35
20.00:
0.89
3.49+ 1.07 155.50'15.65
a Plots B1, A1 and A7 are those of recommended rate ( r . r . 1 of, 10 times of r.r. of the herbicide, and control, respectively. Values are means standard deviation of 6 replicate samples from plots B1 and A7 and of 4 replicate samples from plot A l l respectively. This research is the part of a project, Green Energy Program (GEP-82, 83, 84-111-2-1) financed by the Ministry of Agriculture, Forestry and Fisheries of Japan.
-341-
REFERENCES ISHIKAWA, K., SHINOHARA, R., YAGI, A., SHIGEMATSU, S . , KIMURA, I.: Identification of S-benzyl N,N-diethylthiocarbamate in paddy field soil supplied with benthiocarb herbicide. J. Pesticide Sci. 5: 107-109, 1980. MOON, Y. H., KUWATSUKA, S . : Microbial aspects of dechlorhation of the herbicide benthiocarb (thiobencarb in soils. J. Pesticide Sci. 10: 513-521, 1985a. MOON, Y. H., KDWATSUKA, S . : Factors influencing microbial dechlorination of benthiocarb (thiobencarb) in the soil suspension. J. Pesticide Sci: 10: 523-528, 1985b. NAKAMURA, Y. K., ISHIKAWA, K., KUWATSUKA, S . : Degradation of benthiocarb in soils as affected by soil conditions. J. Pesticide Sci. 2 : 7-16, 1977.