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Populations of Cd-tolerant microorganisms in soilspolluted with heavy metalsMikiya Hiraki aa Environmental Biology Division , National Institute for Environmental Studies , Tsukuba ,305 , JapanPublished online: 04 Jan 2012.

To cite this article: Mikiya Hiraki (1994) Populations of Cd-tolerant microorganisms in soils polluted with heavy metals, SoilScience and Plant Nutrition, 40:3, 515-524, DOI: 10.1080/00380768.1994.10413329

To link to this article: http://dx.doi.org/10.1080/00380768.1994.10413329

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Soil Sci. Plant Nutr., 40 (3), 515-524, 1994

Populations of Cd-Tolerant Microorganisms In Soils Polluted with Heavy Metals

Mikiya Hiraki

Environmental Biology Division, National Institute for Environmental Studies, Tsukuba, 305 Japan

Received August 25, 1993; accepted in revised form November 10, 1993

In order to estimate the effect of heavy metal pollution on soil microorganisms, field surveys were carried out in paddy fields, upland fields, and fallow paddy fields located in two areas with heavy metal pollution in Japan (Izuhara area, where the soil samples contained 0.2-15.0 mg Cd kg-I, 11-696 mg Zn kg-I, 0.8-16.2 mg Cu kg-I; Toyama area, where the soil samples contained 0.2-3.9 mg Cd kg-t, 4.7-146 mg Zn kg-I, 0.5-21 mg Cu kg-I). There was no significant correla­tion between the viable counts of microorganisms and heavy metal content of the soils. The dominance value of the bacteria tolerant to 10 mg kg- I Cd (DID value; D,=log(VCc/VCo), where VC, is the viable count on the plates contain­ing c mg L -I of Cd, VCo is the viable count on the plates without Cd) increased significantly with the increase of the heavy metal content in the paddy fields. However, there was no significant correlation between the DIDD value of the bacteria or the DID value of the fungi and the heavy metal content of the soils. The DIDD value of the bacteria tended to be lower in paddy field soils than in upland or in fallow paddy field soils. In a polluted area, the D5DD value of the fungi was higher in the fallow paddy field soils than in the paddy or upland field soils. These results suggest that the effects of heavy metal pollution on soil microorganisms vary depending on the land use.

Key Words: bacteria, fallow paddy field, fungi, paddy field, upland field.

515

It is generally recognized that heavy metals may impair biological processes in eco­systems. Since soil pollution problems due to these metals are becoming increasingly serious, it is important to assess the deleterious effects of the metals on soil ecosystems. A large number of laboratory and field studies have been carried out on the effects of heavy metals on soil microorganisms, which play an important role as decomposers in soil ecosystems (Baath 1989). A field survey on arsenic (As)-polluted fields conducted by the author showed that As-tolerant fungi became predominant in an As-polluted paddy field, while, the effect of As-pollution on soil microorganisms in an upland field differed from that in a paddy field (Hiroki 1993). This observation suggests that the effects of heavy metals on soil microorgan­isms in agricultural fields depend on the land use and method of cultivation, which should be assessed when considering the effects of heavy metals. The present study was designed to analyze the relationship between the heavy metal contents and the microbial popUlation in soils of upland fields, paddy fields, and fallow paddy fields polluted with heavy metals.

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516 M. HIROKI

Table 1. Properties of soil samples collected from the Izuhara area.

No. Land-

W.C b pH CECc T-Cd T-Nd T-Cde T-Zne T-Cue Ex-Cd' Ex-Zn' Ex-Cu'

use" (H 2 O)

IZI P 0.462 6.2 18.3 27 1.8 16.7 1,436 44.0 14.12 253.9 14.0 IZ2 P 0.289 5.7 20.6 31 2.0 7.24 1,280 56.4 6.69 124.8 16.2 IZ3 FP 0.216 6.1 16.5 21 1.3 6.03 1,238 42.8 5.72 93.9 8.6 IZ4 P 0.351 5.7 14.3 14 \,1 0.64 396 26.8 0.73 20.9 7.7 IZ5 U 0.084 4.7 22.7 21 1.4 1.33 534 34.3 0.97 37.2 2.9 IZ6 U 0.162 6.7 31.2 35 2.1 20.3 2,830 92.5 15.00 567.4 0.8 IZ7 U 0.219 6.7 31.0 50 3.2 10.6 3,000 73.0 8.23 695.6 \.2 IZ8 U 0.079 5.1 22.6 38 2.5 3.68 671 48.3 0.22 24.4 4.9 IZ9 P 0.422 6.6 20.6 14 1.5 0.53 303 105.9 0.41 18.2 10.9 IZIO U 0.087 6.2 18.2 21 \.8 0.35 233 48.9 0.45 1 1.4 14.4

" P, paddy field; FP, fallow paddy field; U, upland field. b Water content in wet soil (kg kg-I). C cmo1( +) kg-I d g kg-I. e Total content determined by HCIO.-HN03 -H 2 SO. digestion method (mg kg-I). r Content of extractable form determined by 0.1 mol L -1 HCI (mg kg-I).

STUDY SITES AND METHODS

The field survey was carried out in two areas in lapan, one in Izuhara City, Nagasaki Prefecture (abbreviated as Izuhara area), and the other in Toyama City and Fuchu City, Toyama Prefecture (abbreviated as Toyama area).

The Izuhara area had been polluted with irrigation water from the Sasu River and Shiine River containing cadmium (Cd) and other heavy metals, in the upstream catchment where which a zinc (Zn) and lead mine is located (Aomine and Honjo 1954). The soil types in the study area were Fluvisols and Yellow soils. The field survey was carried out on Sep. 8, 1982. Soil samples were collected from a total often plots including paddy fields (IZ1, 2, 4, 9), upland fields (IZ5, 6, 7, 8, 10), and a fallow paddy field (IZ3). Two plots on upland fields (IZ6, 7) were contaminated with slag from the mine, while two plots upstream of the mine (IZ9 and 10) were not polluted (Table I).

The Toyama area is located in the downstream part of the catchment of the lintu River, in the upstream catchment of which the Kamioka zinc mine is located. The irrigation water from the river had been polluted with Cd and other heavy metals. Soil types in the study area were Fluvisols and Andosols. Field surveys were carried out on May 9, 1984, Nov. 13, 1984, and Nov. 13, 1985. Soil samples were collected from paddy fields (5 plots of Andosols and 8 plots of Fluvisols), upland fields (2 plots of Andosols and 3 plots of Fluvisols), fallow paddy fields (5 plots of Andosols and 7 plots of Fluvisols) and a garden field (Fluvisol). Among these plots, two paddy fields (TAl3 and TSI4) and the garden plots (TS22) had not been irrigated with the polluted water (Table 2).

In both study areas, a soil sample was collected from the surface (0-5 cm) soil of each plot and sieved (2-mm mesh) to count the microbial population as well as determine the contents of total and exchangeable heavy metals and total carbon (T -C). The counts of the micro bial population were carried out as soon as possible, otherwise the soil samples were kept at SOC until use.

To analyze the population of microorganisms, viable counts (VC) of bacteria and fungi were performed using the dilution plate-count technique (Clark 1965; Menzies 1965). Bacteria were cultured on an albumin agar medium plate consisting of egg albumin 0.25 g, glucose 1.0 g, K2 HP04 0.5 g, MgS04 • 7H 20 0.2 g, trace of Fe2 (S04)3, and agar 15.0 g per liter,

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Cd-Tolerant Microorganisms in Polluted Soils 517

Table 2. Properties of soil samples collected from the Toyama area.

No. Land- pH usea W.C b (H

2

0) CECc T-Cd T-Nd T-Cde T-Zne T-Cue Ex-Cd' Ex-Zn' Ex-Cu'

Andosols TAl P TA2 P TA3 FP TA4 FP TAI2 FP TA13 P TA26 P TA27 U TA28 FP TA29 P TA30 FP TA31 U

Fluvisols TS5 P TS6 FP TS7 FP TS8 P TS9 P TSIO U TSII FP TSI4 P TSI5 FP TSI6 FP TS17 U TS18 P TSI9 FP TS20 P TS21 FP TS22 G TS23 P TS24 U TS25 P

0.495 6.0 0.519 6.1 0.403 5.7 0.385 5.7 0.365 6.6 0.434 5.7 0.562 6.1 0.413 5.9 0.389 5.6 0.482 6.3 0.418 6.1 0.413 5.8

0.323 6.1 0.371 6.5 0.277 5.6 0.470 5.9 0.450 5.7 0.243 4.7 0.279 6.3 0.279 6.0 0.254 5.3 0.393 5.4 0.359 6.3 0.459 6.9 0.366 5.6 0.511 6.3 0.296 5.9 0.229 5.7 0.321 6.2 0.303 6.4 0.325 6.4

33.7 35.1 45.8 41.5 17.2 32.8 29.8 27.3 32.0 30.9 34.7 25.9

9.4 9.2

16.8 12.1 9.1 7.1

11.6 15.8 10.3 28.9 34.2 28.5 31.6 27.4 19.7 22.8 18.5 9.4

10.1

66 71 63 58 38 60 60 48 41 45 60 56

22 24 22 26 18 16 18 20 23 26 23 23 31 28 17 14 20 14 18

5.5 6.4 5.3 4.8 3.4 5.2 5.1 4.1 3.5 3.9 4.9 4.3

2.2 2.4 2.0 2.5 1.6 2.0 1.7 1.9 2.0 2.5 2.2 2.2 2.3 2.5 1.4 1.0 1.7 1.4 1.6

2.35 395 3.53 412 2.30 354 3.18 527 4.41 859 0.77 122 2.98 519 2.03 429 2.29 472 2.29 552 2.23 457 2.19 411

0.57 203 0.71 299 2.07 678 1.36 617 0.95 320 0.65 238 2.78 923 0.14 83 1.08 351 0.96 170 0.75 156 0.72 128 0.93 164 0.62 123 1.15 385 0.38 79 1.11 361 1.07 400 1.23 549

30.8 2.32 99.2 1.13 34.6 3.08 93.1 1.19 35.1 2.12 99.1 1.46 42.5 2.89 89.8 2.29 50.7 3.94 98.6 6.41 21.4 0.62 16.9 0.46 37.0 2.71 96.2 3.45 38.7 1.95 95.2 2.74 32.6 2.23 87.2 3.01 34.7 2.29 78.5 4.75 42.2 2.11 80.2 3.90 34.5 2.1080.5 2.17

17.3 0.55 36.0 5.44 22.4 0.72 44.3 6.57 38.6 1.76 100.8 12.75 36.6 1.07 78.6 10.62 27.7 0.70 48.1 7.51 21.8 0.54 35.3 6.13 53.3 2.30 146.3 21.17 16.6 0.20 11.2 3.89 27.5 1.00 57.6 7.43 21.3 0.66 31.7 4.47 23.5 0.56 17.5 5.68 18.1 0.50 13.1 4.67 25.2 0.68 32.9 3.21 18.3 0.47 14.5 4.57 24.6 0.75 47.6 5.61 15.5 0.19 4.7 1.59 23.4 0.87 47.7 7.27 24.9 0.78 45.3 7.31 28.9 0.99 60.5 10.29

a P, paddy field; FP, fallow paddy field; U, upland field; G, garden soil. b Water content in wet soil (kg kg-I). C cmol( +) kg-I. d g kg-I. e Total content determined by HCIO.-HN03 -H2 SO. digestion method (mg kg-I). f Content of extractable form determined by 0.1 mol L -1 HCI (mg kg-I).

adjusted to pH 6.9, at 28°C for 7 d, and the fungi were cultured on a Martin's rose Bengal agar medi urn plate consisting of KH 2 P04 1.0 g, MgS04 • 7H 2 0 0.5 g, peptone 5.0 g, glucose 10.0 g, rose bengal 0.033 g, and agar 20.0 g per liter, adjusted to pH 6.8, at 2YC for 4 d. To count the number of Cd-tolerant microorganisms, a CdCl2 solution was added to these media at Cd concentrations of 10, 100, and 500 mg Cd L -1. The dominance value of the Cd-tolerant microorganisms was calculated by the following formula.

De, dominance value of microorganisms which are tolerant to c mg L -) of Cd; VC e, viable count on the plates containing c mg L -1 of Cd; VCo, viable count on the plates without Cd.

T -C content was determined with a CHN analyzer (Carlo-Erba, model 1106). The total content (T-) of Cu, Zn, and Cd was determined using the HN03-H 2S04 -HCI04 digestion

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518 M. HIROKI

method followed by atomic absorption spectrometry (Shimadzu, AA 640-12) for Cu and Zn or by flameless atomic absorption spectrometry (Shimadzu, GFA-2/ AA640-12) for Cd.

The amounts of exchangeable (Ex-) Cu, Zn, and Cd were determined using the 0.1 mol L -I HCI extraction method followed by atomic absorption spectrometry as well as the determination of the total heavy metal content.

A grass sample was collected from one of the plots of an Andosol in the Toyama area (T A30), which was a polluted fallow paddy field. The sample was washed in distilled water to remove soil particles, air-dried and then separated into shoots and roots to determine the heavy metal content of each part. The metal content was determined by HN03-HCI04

digestion in a double vessel digestion bomb (Okamoto and Fuwa 1984) followed by inductively coupled plasma atomic emission spectrometry (Jarrel-Ash, ICAP-7S0).

RESULTS

Heavy metal contents in the study areas In the Izuhara area, the soil samples from slag-contaminated plots (IZ6 and IZ7)

contained high levels of Ex-Zn (567 and 696 mg kg-I) and Ex-Cd (1S.0 and 8.2 mg kg-I); samples from unpoll uted plots (IZ9 and IZ 10) contained about 18 mg kg-lor less Ex-Zn and about O.S mg kg-I soil or less Ex-Cd. The Ex-Cu content of the samples was about 1-16 mg kg-I (Table I). There was a positive correlation (r= 0.74S*) between the contents of Ex-Zn and Ex-Cd, but no significant correlation between the contents of Cu and the other heavy metals (Table 3).

In the samples of Fluviso1 soil from the Toyama area, the contents of Ex-Cu ranged from 1.59 (TS22, non-polluted garden) to 21.2 mg kg- 1 (TSll, polluted fallow paddy field), Ex-Zn from 4.7 (TS22) to 146 mg kg-I (TSIl), and Ex-Cd from 0.19 (TS22) to 2.30 mg kg-I (TS II); in the Andosol samples the Ex-Cu contents ranged from 0.46 (T A 13, non-polluted paddy field) to 6.41 mg kg- 1 (TAI2, polluted fallow paddy field), Ex-Zn from 16.9 (TAl3) to 99.2 mg kg-l (TAl, polluted paddy field), and Ex-Cd from 0.62 (TA!3) to 3.94mgkg-1 (TA 12) (Table 2). In each soil type, there were positive correlations (r= 0.S92*-0.976**) among the exchangeable contents of the three heavy metals, except for one case between Ex-Cd and Ex-Cu from the Toyama Andosol (Table 3).

Microbial population in the study areas There was no significant relationship between the Ex-Cd content and the viable counts

(YCas) of bacteria (Fig. 1) or fungi (Fig. 2) in the soil samples, nor was there any significant correlation between the YCas of the microorganisms and the contents of heavy metals or T-C.

Table 3. Correlations among the exchangeable contents of Cd, Zn, and Cu in the soil samples.

Study area

Ex-Cd-Ex-Zn Ex-Cd-Ex-Cu Ex-Zn-Ex-Cu

lzuhara Toyama (Andosols) Toyama (Fluvisols) (n= 10) (n= 12) (n= 19)

0.745' -0.107 -0.527

0.749** 0.592' 0.336

0.976" 0.950" 0.959"

Values followed by • or •• are significant at the 5% or I % level.

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Cd-Tolerant Microorganisms in Polluted Soils 519

20 20 20 (a) (b) (c)

";' be 0

0 ..14

~ 0 ~ u

0 10 10 0 10 ~O Do of ......

0 ~ 0 0

0 0 0 '': .. • .. ,p OJ 0

0 ·0 ... o 0 0 0 u ~ 0 ~ 0 I:Q 0

0

10 20 00 2 3 4 00 2 Cd content (mg kg-I)

Fig. 1. Relation between Cd-content and number of bacteria in soil. (a) lzuhara area; (b) Toyama area (Andosols); (c) Toyama area (Fluvisols) . • , paddy field; 0, upland field: 0, fallow paddy field; 6, garden soil.

8 8 8 0 (a) (b) (c)

";' 0 be 6 6 0 6 ..14 0

::J 0 0

~ U Do 0

"'0 4 0 4

0 0 0 ...... 0 0

'5"0 0 A ~ "" 0 0 c 0 2 2 00 0 0 0 ::l .. • p... 0 0 ..

iii 0 0

00 10 20 00 2 3 4 00 2 Cd content (mg kg-I)

Fig. 2. Relation between Cd-content and number of fungi in soil. (a) lzuhara area; (b) Toyama area (Andosols); (c) Toyama area (Fluvisols) . • , paddy field; 0, upland field; 0, fallow paddy field; 6, garden soil.

Predominance of Cd-tolerant microorganisms In the Izuhara area, the D lo values of the bacteria increased with the increase of the

Ex-Cd content (Fig. 3a), whereas in the Toyama area, such a relationship was observed only in the paddy fields (Fig. 3b, c); there were significant correlations between the Ex-Cd content and the D lo values of bacteria in the paddy field samples in the Toyama area (Andosol, r=

0.943*; Fluvisol, r = 0.860* *), but no significant correlation for the fallow paddy fields. In the case of bacterial DlOo and D500 values, there were no distinct relationships between the heavy metal content and the Dc values of the samples from either study area (Figs. 4, 5). In the case of the fungi, the relationship between the heavy metal content and the D IO , D lOo , or D500 values was significant in only one case, the D500 value in the Toyama Fluvisol (Figs. 6-8).

Cd-tolerant microorganisms in paddy field soils and other soils In the Izuhara area, the DIOO and D500 values of the bacteria in the samples of paddy field

soils tended to be lower than those in the upland field samples (Figs. 4a, 5a), whereas there was no such difference for the bacterial D lo values (Fig. 3a). In the Toyama area, the D lo ,

D loo , and D500 bacterial values were higher in the fallow paddy field samples than in those

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520

.~ .... ~ u <1l .c ..... 0

'" cu ;l

til > 0

Cl~

t (a)

• 0 .. 0

r=0.637"

.. ' 0

0

M. HIROKI

(b)

-1

......•

.•..... •••••• r=0.943·

•••. 0

.' .," 0

_1L---~-----L----~--~ _1L-~~ ____ ~~ __ ~ __ ~ o 10 20 0 1 2 3 4

Cd content (mg kg·1 )

-1

(c)

r8 '0 ?~ · ," l

./ .... : r-0.860"

, .. '. o

o o

Fig. 3. Relation between Cd-content in soil and the dominance value of Cd-tolerant bacteria (D,o). (a) Izuhara area; (b) Toyama area (Andosols); (c) Toyama area (Fluvisols). II, paddy field; 0, upland field; 0, fallow paddy field; l>, garden soil. Lines represent the linear relationship between the Cd-content and the D,o value calculated from all of ten samples (a) or from five and seven samples of paddy field (b, c).

-t -1 -1

(a) (b) (c) <1l 'r: 0 cu 0 .... u 0 0 0 0 <1l .c

~ 0

..... 0 • "-.. 0

0 0 0

'" -2 -2 • 0

0 • cu 0 • ;l

" • til " > 0 • • 0 0 ~ " '" .. " Cl

" 0

-20 10 20 -3 0 2 3 4

-30 2

Cd content (mg kg·1 )

Fig. 4. Relation between Cd-content in soil and the dominance value of Cd-tolerant bacteria (DlOo)' (a) lzuhara area; (b) Toyama area (Andosols); (c) Toyama area (Fluvisols) . • , paddy field; 0, upland field; 0, fallow paddy field; l>, garden soil.

-1 -2 -2 (a) B (b) 0 (c)

<1l 0 0

0 0 0 0 ;::: cu 0 "- .cP 0 .... u -2 -3

" -3

<1l 0 0 • .c 001 ..... 0 0 0 • • 0

'" cu • • ;l -4 • -4 • • til -3 0

• > • • 0 • 0 • '" Cl

-4 -5 -5 0 10 20 0 1 2 3 4 0 2

Cd content (mg kg·1 )

Fig. 5. Relation between Cd-content in soil and the dominance value of Cd-tolerant bacteria (D50o)' Ca) Izuhara area; (b) Toyama area (Andosols); (c) Toyama area (Fluvisols) . • , paddy field; 0, upland field; 0, fallow paddy field; l>, garden soil.

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Cd-Tolerant Microorganisms in Polluted Soils 521

(a) (b) 0 (c)

0.2 0.2 0.2 0

.0. 0 co t:: 0

.2 0.0 0.0 B 0.0 0 • • 0

0 .... • 0 • 0 0

0 0 0 • II

II)

-0.2 A 0 Oil. C1J -0.2 -0.2 • ;l

OJ 0 • .. > • • • :3 -0.4 -0.4 • -0.4 0 0

-0.60 10 20-0.6 0 1 2 3 4-0.6 0 2

Cd content (mg kg-1 )

Fig. 6. Relation between Cd-content in soil and the dominance value of Cd-tolerant fungi (D,o). (a) lzuhara area; (b) Toyama area (Andosols); (c) Toyama area (Fluvisols) . • , paddy field; 0, upland field; 0, fallow paddy field; "'-, garden soil.

-0.2 (a) 0.0 (b)

0.0 (e)

0 • 0 0 • . 0. -0.4 0

t:: • .2 • -0.4 0 -0.4 • i 0

...... 0 II)

C1J

.. • 0 0

6· o· A &J 0 ;l ..

OJ > 0 -0.8 0 0 :3

0 -0.8 .. 0 0

-0.8

0 0 • • -1.0 L..._~ __ -'-__ ~ __ --' -1.2 -1.2 L..._~_--"-_~ __ -'---_~

0 10 20 0 1 2 3 4 0 2 Cd content (mg kg-1 )

Fig. 7. Relation between Cd-content in soil and the dominance value of Cd-tolerant fungi (DlOo). (a) Izuhara area; (b) Toyama area (Andosols); (c) Toyama area (Fluvisols). It, paddy field; 0, upland field; 0,

fallow paddy field; "'-, garden soil.

-2 0 0 0 (a) (b) ee)

0

0 0 .0.

8 0

0 I':: -1 -1 0 ;l -3 0 '?f1 ..... 0 .... • • ..

0 • .. II)

-2 -2 • 0 C1J 0 ;l 0 • OJ

0 A

• • > -4 0 • 0 • 0 -3 -3 g 0 on 0 0

• -50 10 20

-4 -4 0 2 3 4 0 2

Cd content (mg kg-1 )

Fig. 8. Relation between Cd-content in soil and the dominance value of Cd-tolerant fungi (D50o). (a) Izuhara area; (b) Toyama area (Andosols); (e) Toyama area (Fluvisols) . • , paddy field; 0, upland field; 0, fallow paddy field; "'-, garden soil.

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522 M. HIROKI

of the paddy fields, but there was no significant difference between the values in the paddy field samples and those in the upland field samples (Figs. 3b, 4b, 5b, Figs. 3c, 4c, 5c). In both study areas, the DID' DIDO' and D500 values of the fungi ranged from -0.43 to 0.25, from .- 1.085 to - 0.138 and less than - 0.423, respectively. There were no significant differences among the values in the paddy field samples, those in the upland field samples and those in the fallow paddy field samples (Figs. 6, 7), whereas the D500 values of the fungi varied with the area and land use. The D500 values of the fungi in the Izuhara area were lower than those in the Toyama Fluvisols and Andosols; in the samples from fallow paddy field soils in the Toyama area the D500 values of the fungi ranged from -1.39 to -0.42, whereas the values from the samples from polluted paddy and upland fields and from unpolluted fields were lower (Fig. 8).

Heavy metal content in grass on a fallow paddy field polluted with heavy metals The heavy metal contents in a grass (Digitaria ciliaris (Retz.) Koel.) collected from a

fallow paddy field polluted with heavy metals on the Toyama Andosol (T A30) were 309 mg kg- I Zn, 19.9 mg kg- I Cd, and 9.0 mg kg- I Cu in roots, and 288 mg kg- I Zn, 1.2 mg kg- I Cd, and 51.5 mg kg- I Cu in shoots. The content of Cd in the roots was about 9 times the Ex-Cd content of the soil from which the plant sample had been collected, and the contents of Zn in the roots and Cu in the shoots about 4 and 13 times as high as the values of the respective exchangeable heavy metals in the soil.

DISCUSSION

A previous study by the author (Hiroki 1992) showed that the number of bacteria and actinomycetes decreased markedly with the increase of the heavy metal contents in soils polluted with heavy metals along the Watarase River, whereas in the present study no significant correlations between the numbers of soil microorganisms and heavy metal contents were revealed. In the current study areas the T -Cu content in soil was higher than the average value for Japanese farm land soil (mean, 24.82 mg kg-I; median, 25.50 mg kg-I; Environment Agency 1984); these values were, however, lower than those recorded in the polluted soils only the Watarase River (300-750 mg kg-I). The lower content of Cu in the soils of the study areas may account for the fact that the microbial numbers were not affected significantly by heavy metal pollution, in spite of the high T-Zn content (Izuhara, 3,000 mg kg-I; Toyama, 920 mg kg-I).

Although distinct effects of heavy metal pollution on microbial populations were not observed, the bacterial DID values increased with the increase of the heavy metal contents in the paddy field soils. Although this observation suggests that the increase of the content of heavy metals inhibits the growth of Cd-sensitive bacteria, it is possible that the pollutant inhibiting the growth of Cd-sensitive bacteria was not Cd, because the predominant Cd-tolerant bacteria may be tolerant to other heavy metals (Duxbury and Bicknell 1983).

There were no significant correlations between the DIDO or D500 values of the bacteria and the content of heavy metals, whereas there was a distinct difference between the D500

values of the bacteria in samples from paddy field soils and the values in the upland or fallow paddy field samples, the former being lower than the latter. It is possible that the toxicity of Cd and other heavy metals to microorganisms is suppressed under the reducing conditions of the paddy field soils, since the heavy metals precipitate as sulfide under reducing conditions. Also the amount of Cd taken up by rice plants may be higher in paddy

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Cd-Tolerant Microorganisms in Polluted Soils 523

field soils than in upland field soils. If so, bacteria which are not tolerant to Cd may be able to grow more easily in Cd-polluted paddy field soils than in polluted upland field soils, and the dominance value of the Cd-tolerant bacteria may be lower in the paddy field soils than in the upland field soils. However, there were no significant correlations between the D500

values of the bacteria and the contents of heavy metals in the soils, and the D500 values of the bacteria were actually higher in the unpolluted upland field soil (IZIO) and the garden soil (TS22) than in the polluted paddy field soils. This fact suggests that the higher D500

values in upland field soils than in paddy field soils, regardless of the amount of heavy metal pollutants, may be ascribed to another factor. Mechanisms of tolerance to heavy metals of bacteria vary (Wood and Wang 1983), and it is possible that the mechanism of tolerance to high concentrations of Cd is different from that to low concentrations, the former being specific to the aerobic bacteria which do not occur in paddy field soils.

The dominance values of the Cd-tolerant fungi (Dlo , from -0.43 to 0.25; D loo , from -1.085 to -0.138; D50o , < -0.423) were higher than those of the bacteria (D IO , from - 1.86 to -0.192; D loo , from -2.96 to 0.95; D 50o , <-1.25). These data are in agreement with the observations in the Cu-polluted soils among the Watarase River (Hiroki 1992), and the high tolerance of fungi to heavy metals may account for the fact that there were no significant correlations between the dominance values of the Cd-tolerant fungi and the contents of heavy metals in the soils. Yamamoto et al. (1981), who surveyed microbial populations in Cu-polluted soils, showed that the Cu-tolerant fungi became predominant in soils polluted with a high level of Cu, whereas the presen.t study did not reveal any significant correlations between the number or dominance value of the Cd-tolerant fungi and heavy metal contents. The effects of Cd and Zn pollution on the soil microbial population may be different from those of Cu pollution.

The availability of Cd in paddy field soils was restricted under reducing conditions. This may be one of the reasons why the D500 values of the fungi in the fallow paddy field soils were higher than those of the paddy field soils polluted with the same amount of Cd as that in the fallow paddy field soils, in the Toyama area (Andosols and Fluvisols). However, the predominance of fungi which are tolerant to a high concentration of Cd was not observed in the polluted upland field soils in the Toyama area (T A27, TS 10, TS 17) and in the lzuhara area. It is possible that the predominance of the Cd-tolerant fungi in the fallow paddy field soils in the Toyama area may be due to other causes.

In a fallow paddy field (T A30) where Cd-tolerant fungi were predominant, grasses took up large amounts of heavy metals. For the microorganisms which decompose grasses containing a large amount of heavy metals, it would be advantageous to be tolerant to the heavy metals. If so, microorganisms tolerant to heavy metals may predominate in the soils of the polluted fallow paddy fields.

Acknowledgments. The author wishes to thank Dr. T. Asami, Professor, lbaraki University, for the collection of soil samples from the Toyama area, Mr. A. Otake, Toyama Prefectural Office, and Mr. T. Nishimura, agricultural counselor of Nagasaki Prefecture, for their help in the field survey, and Dr. M. Nishikawa, National Institute for Environmental Studies, for measuring the contents of heavy metals by ICP.

REFERENCES

Aomine, S. and Honjo, Y. 1954: On the soils affected by mines in Tsushima. Jpl1. J. Soil Sci. Plant Nutr., 25,27-30 (in Japanese with English summary)

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at 0

0:11

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524 M. HIROKI

Baath, E. 1989: Effects of heavy metals in soil on microbial processes and populations (a review). Water Air Soil POl/Zit., 47, 335-379

Clark, F.E. 1965: Agar-plate method for total microbial count. In Methods of Soil Analysis, Part 2, Ed. CA. Black, D.O. Evans, J.L. White, L.E. Ensminger, and F.E. Clark, p. 1460-1466, American Society of Agronomy, Madison, Wisconsin

Duxbury, T. and Bicknell, B. 1983: Metal-tolerant bacterial populations from natural and metal-polluted soils. Soil Bioi. Biochem., 15, 243-250

Environment Agency 1984: Oojo Osen Kankyo Kijlln Settei Chosa, p. 22, Environment Agency, Tokyo (in Japanese)

Hiroki, M. 1992: Effects of heavy metal contamination on soil microbial population. Soil Sci. Plant Nutr., 38, 141-147

Hiroki, M. 1993: Effect of arsenic pollution on soil microbial population. Soil Sci. Plant Nutr., 39, 227-235 Menzies, J.~. 1965: Fungi. In Methods of Soil Analysis, Part 2, Ed. CA. Black, D.O. Evans, J.L. White, L.E.

Ensminger, and F.E. Clark, p. 1502-1505, American Society of Agronomy, Madison, Wisconsin Okamoto, K. and Fuwa, K. 1984: Low-contaminated digestion bomb method using a Teflon double vessel

for biological materials. Anal. Chem., 56, 1758-1760 Wood, J.M. and Wang, H. 1983: Microbial resistance to heavy metals. Environ. Sci. Technol., 17, 582-590 Yamamoto, H., Tatsllyama, K., Egawa, H., and Furuta, T. 1981: Microfiora in soils polluted by copper mine

drainage. J. Sci. Soil Manure, Jpn., 52, 119-124 (in Japanese)

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