natural fluctuation of microbial activity related to

J. Pesticide Sci. 24, 386-392 (1999)

Original Article

Natural Fluctuation of Microbial Activity Related to Nitrogen

Cycle in Rice Paddy Soils as a Basis for Assessing the Side-Effect

of Pesticides on Soil Ecosystem

Yuko OKAMOTO, * Kousuke SUYAMA, *Kazuhito ITOH, *Miho ITAMOCHI,

Yasuhiro KAGAwA, *Shinji KAJIHARA, *Takahiro IKUSHIMA, *Hitomi MIYAMOTO*

and Hiroki YAMAMOTO

*Faculty of Life and Environmental Science, Shimane University,

Matsue 690-8504, Japan *Agricultural Products, DuPont K. K., A rco Tower, 8-1, Shimomeguro 1-chome,

Megzro-ku, Tbkyo I53-0064-Japan

(Received April 16, 1999; Accepted July, 23, 1999)

Soil microbial parameters of two different rice paddies were monitored, from May 1997 to Dec. 1998

(20 months). During the two growing seasons, the natural variability in the microbial communities activity were monitored in order to assess the effects of pesticide usage on rice paddy soils. The study focused mainly on the effect of pesticides on the nitrogen cycle such as nitrification, ammonification,

protease activity and ammonium oxidizing bacterial population (AOB) being what was monitored. The coefficient of variation(CV) was the statistical tool used to monitor the variability in the study. During the two growing seasons, the CV for nitrification, ammonification, protease activity and AOB were 16-20, 39-53, 31-62 and 88-174%, respectively, which reflects the fluctuation caused by the natural stresses as well as anthropologic stresses. These results were much wider than the fluctuation observed in the short period or in laboratory studies and should be used as reference when conduct risk assessments of pesticides to the microflora in rice paddy soil.

Key words: nitrification, ammonification, protease activity, ammonium oxidizer, risk assessment, long-term monitoring.

INTRODUCTION

There is increasing concern about the side-effects of

pesticides in the environment, and a number of papers have been published on the side effects of pesticides. ), 2) The side-effects on soil microflora are one of the area of concern in recent years especially in Europe. The regulatory guidelines have been developed by SETAC, 3 OECD4 and other international organizations. 5 In Japan, there is also concern over the side effects of

pesticides on the microflora and Yamamoto et al. 6 have developed recommended test guidelines. Therefore the regulatory requirements on the risk assessment to the effects of pesticides on soil microorganisms will be added in the near future. Currently, most of the papers pub-lished thus far on the subject of side-effects of pesticides on the soil microflora are for test methods, and the discussion from these papers focus little attention on the interpretation of the results.

Microorganisms are of prime importance in the recy-cling of key elements essential for biological processes and thus for the maintenance of soil fertility. Since current agricultural practices apply biologically active compounds to soil as a part of pests control, it is impor-tant to carefully understand the effects of these com-

pounds on the soil microbial community. It is, how-ever, crucial that any such artificially induced effects be considered in the context of effects induced by natural stresses. Most of studies concerning with pesticide effects on soil ecosystem are just laboratory studies. Is a high degree of statistically significant difference in a laboratory study regarded as an environmentally significant difference? The magnitude of microbial responses to chemical stress should be compared to those

produced by natural stress factors. From ecological data of fluctuations of temperature, water potential, pH,

physical disturbance of soil, reduced gas exchange, it can be concluded that microbial population depressions of 50

Journal of Pesticide Science 24 (4) November 1999 387

to 90% occur under natural field conditions.7) Only a few studies have reported the natural fluctuations of microflora from such a perspective. ') The purpose of this study is to develop the reference for conducting a risk assessment of the effects of pesticides on soil ecosystem from the perspective considering natural fluctuations.

In this report, seasonal fluctuations of the microbial

parameters relating to the nitrogen cycle in actual rice paddy fields will be discussed. Other parameters consid-ered to be useful criteria to assess potential side effect of

pesticides to soil microorganisms such as microbial biomass and population measured at the same time will be reported following this report. The result shown in this report will provide useful information as reference to conduct risk assessment on the side-effects of pesticides to soil microorganisms in rice paddy fields, since the results include the possible rice herbicide effect in the context of effects by the natural stresses.

MATERIALS AND METHODS

1. Test Paddy Sites for Long Term Studies Two different rice paddies regarding to the chemical

inputs were used to determine possible difference in response of soil microflora to natural stress that might have been caused by the use of various pesticides and/or chemical fertilizers for long years. One paddy had no input of chemicals for several years while the other was in the same general area and used for conventional rice cultivation including the use of pesticides and chemical fertilizers. These rice paddies are located in the same watershed of the upper Kososhi stream located in Matsue city, Shimane prefecture. Rice paddy A (18a, no chemi-cals) locates upper area of the stream (Fig. 1), and has been received no pesticide or chemical fertilizer for more than 5 years. In this rice paddy, hand weeding was conducted twice in June. The other rice paddy B (23a, conventional) locates about 800 meters lower stream from paddy A, and has been employed conventional rice cultural practice that includes the use of various chemi-cals.

Soil microflora in these paddy fields were monitored

from May 1997 to Dec. 1998. During May to Septem-ber, soil sample was collected for 2-7 times per month and in the rest of the period sampling was conducted about once in a month. The work put into paddy A (no chemical) and chemicals (pesticides and fertilizers) put into paddy B (conventional) in addition to common

practices with actual date of input are shown in Table 1. The climatic data such as rainfall, atmospheric temper-

ature, paddy water temperature and soil temperature were measured in the field or collected from the public data base. Atmospheric and soil temperature at the sampling times in 1998 were as below; atmospheric tem-

perature were 6. 0-31. 2C (av. 19. 9C) in paddy A and 4. 9-29. 5C (av. 18. 8C) in paddy B, soil temperature were 4. 5-27. 0C (av. 18. 1C) in paddy A and 4. 9-25. 5C (av. 17. 5C) in paddy B. According to these parameters, the area used for this study is considered as a typical rice

paddy field in south west region of Japan. The rice growing practices in both paddies were about the same pattern and the climatic condition was also the same.

2. Soil Sampling The surface paddy soil (gray low land soil) was col-

lected using plastic column core sampler (300 mm X 55 mm i. d.) at the test sites. Twenty soil columns from each

paddy were collected at one time. The columns were brought back to the laboratory within one hour and columns were kept straight up in order not to disturb soil cores. Each soil column was immediately divided into three layers, upper 0-2, lower 2-10 cm and the rest. After dividing each soil column into layers, each of the top two layer collected from the individual paddies were mixed thoroughly. Hereafter, 0-2 cm layer sample is called "upper layer soil" and 2-10 cm "lower layer soil". The soil samples were added with distilled water to be slurry and were sieved through 2 mm mesh and used for further studies. Water contents of the slurry was around 50%. Soil samples were used immediately after the

preparation for the study. The physico-chemical characteristics of the soils in

paddy A and B are shown in Table 2.

3. Microbial Parameters Monitored for the Study There is inadequate knowledge of what a soil eco-

system is and which process and organisms are of major importance in maintaining soil quality. Therefore, the

parameters recommended to be useful for assessing the side effects of chemicals under paddy conditions6,11) such as nitrogen cycle, microbial biomass and algal growth were measured in the series of the study. In this study,

parameters related to nitrogen cycle were used. , 3. 1 Nitrification Two grams of soil slurry was placed into a 300 ml flask

with 56 ml of distilled water and lml of 0. 8% (NH4)2SO4 were added bringing the final concentration to 30 yg-N/

Fig. 1 Location map of study cite in upper Kososhi stream,

Matsue, Shimane, showing paddy A and B.

388 日本農薬学会誌第24巻第4号平成11年11月

ml. The flasks were covered with aluminum foil and incubated at 30°C for 3 weeks. After the incubation, the total weight was adjusted to the initial weight with distilled water and filtrated (ADVANTEC 5B). The filtrate was used to measure NH4+-N by indophenol colorimetry, N02--N by sulfanylamide-naphthylethyle-

nediamine colorimetry and N03--N by Cataldo method, respectively. Nitrification activity was calculated from the amount of NO2--, NO3-- and NH4+-N. All the treated tests and untreated control were done in triplicate. 3. 2 Ammonification Forty grams of soil slurry along with 10 ml of distilled

water was placed into 6 test tubes (200 mm x 25 mm i. d. ) with the water layer and soil layer 3 and 8 cm in depth respectively. Three of the six test tubes were incubated at 30°C for 4 weeks while the remaining three were extracted with 30 ml of 4N-KC1. The extracted test tubes were sealed (rubber stopper) tightly and placed on a reciprocal shaking (120 rpm) for 30 minutes. After shaking, the mixture was filtered (ADVANTEC 5B), analyzed for NH4+-N by indophenol colorimetry. The test tubes incubated for 4 weeks were individually

filtered and analyzed for NH4+-N in the same way. The difference in the amount of NH4+-N measured before and after the incubation were converted to the amount of NH4+-N per 4 weeks (pg-N/g dry soil/4 weeks).

Table 1 Work and chemical input into paddy A and B.

H : herbicide, F : fungicide, I : insecticide, SC : soil conditioner (microbial agent). * active ingredient(s) in parenthesis.

Table 2 Physico-chemical characteristics of soils in paddy

fields studied.

a phosphate absorption coefficient. b cation-exchange

capacity.


3. 3 Protease Activities For the assays of soil Z-FLase9 and caseinase, we

modified the method of Watanabe-Hayano9 with differences in pH7. 0 phosphate buffer, 2 g of soil sample, 3 hours of incubation and temperature (30C). Two grams of soil slurry were suspended in 18 ml of

phosphate buffer (pH7. 0, 50 mmol/l), 2. 4 ml of benzyloxycarbonyl-L-phenylalanyl-L-leucine solution (2 mmol/ l Z-FL, SIGMA) and 0. 8 ml of toluene in a tightly-capped glass bottle and shaken (120 rpm) at 30C. After the 3 hours incubation, the reaction was stopped with TCA solution. The reaction mixture was filtered

(ADVANTEC 5B), with 2 ml of filtrate being treated with 1 ml buffer solution and l ml of ninhydrin reagent L8500 (Wako pure chem. Co.) and boiled for 15 minutes. After cooling in ice water to room temperature, the absorption at 570 nm was measured by a spectro-

photometer. The Z-FLase activity was expressed as the amount of liberated leucine pmol/g dry soil/sec. All the treatment and untreated control were done in tripli-cate. Caseinase activity was measured in the similar way by replacing Z-FL with casein. Caseinase activity was indicated as its leucine equivalent (pmol/g dry soil/ sec). 3. 4 Nitrifying Bacteria The MPN method was used for the enumeration of

ammonia oxidizing bacteria (AOB). A serial 1: 10 dilution sequence of the soil sample was prepared and 10-2 to 10-6 dilutions were inoculated to the medium containing (NH4)2SO4, 0. 5 g/l; NaCI, 0. 3g/l ; K2HPO4, l. Og/l; MgSO4. 7H2O, 0. 3 g/l; FeSOe7H2O, 0. 03 g/l; CaCO3, 7. 5 g/l. After incubation for 4 weeks at 30C, culture tubes

were checked with diazo-coupling reagent for the positive

growth. 10 The MPN of organisms was estimated with the number of positive tubes by reference to a MPN table.

RESULTS

1. Nitrification Nitrification activity is one of the indicators of soil fertility in regards to supplying nitrogen, essential ele-ment for plant growth. The nitrifying activity of the

paddy soils monitored for 20 months are shown in Fig. 2. As shown in Fig. 2, the fluctuation pattern of the activities in any soil layers were similar. The nitrification activities in both layers were almost the same till winter season and then the activity in upper layers were significantly high during winter time. The activ-ities of both layers in paddy A compared to the activities of paddy B at the same timing were observed to be almost higher. The range of fluctuation in paddy A and B was 347-

831 and 318-903 jig-N/g dry soil/3w, respectively, which is considered not to be significantly different. There was also no significant difference in the range, in

upper and lower layers in both paddy A and B.

2. Ammonification Ammonification reflects the capacity of degrading

nitrogenous material to the ammonium form in soil which is provided to be utilized by plants. Ammonification activities measured in both layers in both paddy A and B are shown in Fig. 3. The fluctuation pattern in upper and lower layers and

fluctuation range was similar in both paddies. The activities in upper layer in both paddies A and B were always significantly higher than that in lower layers through the year and were higher during flooded season. The range of the activity was 20. 9-110 in upper layer and was 2. 4-59. 2 in lower layer.

3. Protease A ctivities The degradation of proteins is an important process in

Fig. 2 Natural fluctuations of nitrification activity monitor-

ed fbom May l997 to Dec. 1998 in upPer(■, ●)and lower

(ロ, ○)layer soil in paddy A(■, □)and B(●, ○).

Fig. 3 Natural fluctuations of ammonification activity

monitored fbom May l 997 to Dec. 1998 in upper(圏, ●)and

lower(□, ○)layer soil in paddy A(■, □)and B(●, ○).

390 日本農薬学会誌第24巻第4号平成11年11月

nitrogen cycle in soils for the efficient utilization and improvement of nitrogen fertilizers. Protease activity in soil has important role in the processli and the activity is considered to be useful parameter to assess the effect of chemicals on nitrogen cycle in soil.

In this study, Z-FLase and caseinase were measured. Z-FLase was measured from Jan. 1998 to Dec. 1998 and caseinase was measured from Sep. 1997 to Dec. 1998. The ranges of annual fluctuation of Z-FLase and caseinase in both rice paddy soil are shown in Fig. 4 and Fig. 5, respectively. Both Z-FLase and caseinase activ-ities were increased after growing season (no rice plant standing) in both paddies and these activities in upper layer were generally higher than in lower layer in both

paddies. There was no significant difference in fluctua-tion pattern between paddy A and B during the monitor-ed period. The fluctuation range of Z-FLase was 111-933 in

upper layers and 63-632 in lower layers, and the range of caseinase was 79-583 in upper layers and 68-471 in lower layers, respectively.

4. Nitrifying Bacteria The seasonal fluctuations of MPN of ammonia oxidiz-

ing bacteria, which oxidize ammonium to nitrite autotro-

phically, in both paddy A and B are shown in Fig. 6. The numbers in flooded period were significantly higher in both paddies A and B. In paddy A, there was no significant differences in the numbers between upper layer and lower layers. On the other hand, in paddy B where conventional rice cultivation was conducted, statistically(t-test) increased numbers of the bacteria were observed in upper layer soil compared to lower layer soil most the year. The fluctuation range in paddy A and B was 0. 10-137

(X 103MPN/g dry soil) in upper layers and 0. 04-15. 5(X 103MPN/g dry soil) in lower layers. From the range of fluctuation, AOB numbers in flooded period in both soil layers were always observed to be higher than drained

period.

DISCUSSION

The natural variability of microbial parameters related to nitrogen cycle measured in the field studies during May 1997 to Dec. 1998 are summarized in Table 3. In the table, range, average, standard deviation(SD) and coefficient of variation(CV) calculated for each parame-ter and separately in four segments (paddy A, B and soil layer upper, lower) are shown. Although the range of fluctuation is different in each

parameter, CV for nitrification was around 20%, for ammonification was around 50%, for protease (Z-FLase and caseinase) activity was around 50% and for AOB

Fig. 4 Natural fluctuations of protease(Z-FLase)activity

monitored from Jan. 1998 to Dec. 1998 in upper(■, ●)and


Fig. 5 Natural fluctuations of protease(caseinase)activity

monitored fbom Sep. 1997 to Dec. 1998 in upper(■, ●)and


Fig. 6 Natural fluctuations of MPN of ammonia oxidizing

bacteria(AOB)monitored from May 1997 to Dec. 1998 in

upper(■, ●)and lower(□, ○)layer soil in paddy A(■,

□)and B(●, ○).


numbers was up to 180%. From these results, the range of fluctuation in continuous 20 month period covering two rice cultivation seasons caused by various effects including natural stress changed widely, however as far as CV concerned, there was not much observed difference in both paddies, nor in upper and lower soil layers. Therefore, CVs obtained in this study are considered as natural fluctuation. The variation of results obtained by laboratory study

on ammonification in paddy soil, for example, in a

previous report12 was 8. 7% of CV in case of control groups. On the other hand, variation for the same parameter in this study was around 50(39-53) % of CV. The difference in variations shows that microbial activ-ities in natural environment are used to fluctuate in wide range. It emphasizes the importance of natural fluctua-tion data to be used as a reference when the ecotox-icological data are interpreted and assessed as risk. There are many reports discussing pesticide effects on

soil microorganisms. Most of the reports, however, discuss the side-effects with just laboratory derived data, and thus do not considered the natural fluctuation in the microbial community nor recovery from the potential effects. 11 Even field studies13, 14) discussed the side-effect of various pesticides on microflora in rice paddy soil with

just statistical significance between treated and control. As described previously, it is critically important to

assess the effects of pesticide in the context of effects induced by natural and anthropologic stress. The varia-bility observed in this study will be useful as a reference

when determining risk assessments of the potential side

effects of pesticides to the soil microflora in the short

period and in the laboratory. The large fluctuations observed in the natural paddy soils in this report will

provide additional information to conduct more realistic, accurate risk assessment for the effects of pesticide on soil

microflora.

The result obtained simultaneously from these two

paddies located in the same climatic area will provide further information on natural fluctuation under different

management practices. Results of this study will pro-

vide useful information in interpreting laboratory data

for field conditions so that more accurate risk assessment

concepts and procedures governing potential side effects

of pesticides on soil microorganisms may be developed.

Table 3 Range, average (Av), standard deviation (SD) and coefficient of variance (CV) calculated for each microbial parameter separately in four segments.

REFERENCES

1) L. R. Anderson: "Pesticide Microbiology," ed. by I. R. Hill & S. J. L. Wright, Academic Press, London, pp. 313-534, 1978

2) S. Kuwatsuka & H. Wada: "List of Literatures on the Effects of Pesticide and Fertilizers on Soil Microorgan- isms, " Japan Soil Association, Tokyo, 1981 (in Japanese)

3) M. R. Lynch: "Procedures for Assessing the Environmental Fate and Ecotoxicity of Pesticides, " SETAC-Europe, Brus- sels, pp. 40-43, 1995

4) OECD Guideline for the Testing of Chemicals, Proposal for a New Guideline 216/217, Soil Microorganisms, DECD,

Paris, 1999 5) H. R. Gerber, J. P. E. Anderson, B. Bugel-Mongensen, D.

Castle, K. H. Domsch, H. P. Malkomes, L. Somerville, D. J.

392 日本農薬学会誌第24巻第4号平成ll年ll月

Arnold, H. Van De Werf, R. Verbeken & J. W. Vonk: Toxicol. & En viron. Chem. 30, 249 (1991)

6) H. Yamamoto, T. Sato, I. Watanabe, A. Katayama, K. Inubushi & K. Senoo: "Recommended Test Guidelines on Pesticide Effects on Soil Ecosystem," A Report of Grant in Aid for Scientific Research (04354006), Japanese Ministry of Education, Culture and Science, 1993 (in Japanese)

7) K. H. Domsch, G. Jagnow & T. H. Anderson: Residue Rev. 86, 65-105 (1983)

8) L. Somerville: "Pesticide Effects on Soil Microflora," ed. by L. Somerville & M. P. Greaves, Taylor & Francis, London,

pp. 5-15, 1987 9) K. Watanabe & K. Hayano: Soil Biol. Biochem. 27, 197

(1995) 10) E. L. Schmidt & L. W. Belser: "Methods of Soil Analysis,

part 2, 2nd ed.," ed. by A. L. Page, R. H. Miller & D. R. Keeney, Soil Sci. Soc. Am. Publisher, Madison, pp. 1027-

1042, 1982 11) K. Watanabe & K. Hayano: Soil Microorganisms 47, 9

(1996)(in Japanese) 12) H. Yamamoto, K. Suyama, K. Itoh & S. R. Sroczynski:

"Reviews in Toxicology 2," IOS press, Amsterdam, pp. 295 -

302, 1998 13) H. Kai, M. Kamata, S. Kawaguchi & K. Kanayama: Jpn. J.

Soil Sci. Plant Nutr. 57, 535 (1986) (in Japanese)

14) M. Kamata, H. Kai, S. Kawaguchi & K. Kawachino: Jpn. J. Soil Sci. Plant Nutr. 58, 517 (1987) (in Japanese)

要約

水田土壌中における窒素循環に関わる土壌微生物活

性の自然変動: 土壌生態系に及ぼす農薬の影響評価

に向けて

岡本悠子, 巣山弘介, 井藤和人, 板持美保, 賀川泰弘

梶原真二, 生嶋隆博, 宮本ひとみ, 山本広基

土壌生態系に及ぼす農薬の影響は, 環境要因による微生

物の数や活性の変動幅との比較の上で評価する必要があ

る. このような観点から, 島根県松江市近郊にある近接し

た肥培管理の異なる2水田を対象に, 1997年5月から1998

年12月までの20か月間にわたって, 土壌中の窒素循環に

関わる微生物活性の自然変動を調査した. 2作期を通じた

硝化活性, アンモニア化成活性, プロテアーゼ活性および

アンモニア酸化細菌数の変動係数(CV)は, それぞれ16～

20, 39～53, 31～62および88～174%であった. これらの値

は肥培管理の違いにかかわらず同程度であり, 室内試験や

短期の圃場試験の結果から土壌微生物に対する農薬の影響

を評価する際に参照されるべきであろう.

natural fluctuation of microbial activity related to

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