E F F E C T O F S I M U L A T E D A C I D R A I N O N T H E G R O W T H O F

S O Y B E A N

YOSHISHISA KOHNO

Biology Department, Abiko Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko City, Chiba 270-11, Japan

and

TAKUYA KOBAYASHI

College of Horticulture, Chiba University, 648 Matsudo, Matsudo City, Chiba 271, Japan

(Received December 30, 1987; revised December 14, 1988)

Abstract. Soybean seedlings (Glycine max) grown in a glasshouse were exposed to simulated acid rain using a solution of deionized water containing sulfate, nitrate and chloride in concentrations and proportions equivalent to those in ambient rain water. Plants were subjected to acid rain treatment twice a week during the growing season, for a 1 hr period at a rate of 5 mm hr -~. When the acid rain was below pH 3.0, visible symptoms developed in the young trifoliate leaves. However, at a pH above 3.0 there was no evidence of visible leaf injury; also tissue dry weights and leaf areas were not affected, even after 7 weeks of exposure. The number of root nodules in plants exposed to acid rain at pH 4.0 tended to be higher than those of control plants maintained at pH 5.6, but decreased subsequently with decreasing pH. Based on our results current ambient levels of rain acidity in Japan should not have an adverse impact on seedling growth in soybean.

1. Introduction

The first evidence o f ac id ra in effects in J a p a n was observed in morn ing glories

(Pharbitis nil) and aza lea (Rhododendron spp.) dur ing the ear ly 1970's, and a p p e a r e d

as d i sco lo ra t ion o f the petals (Kohno , 1984). Since tha t t ime, there have been active

research p r o g r a m s in J a p a n on acid depos i t ion . Recent ly , Sekiguchi et al. (1986)

r epo r t ed tha t d i eback o f J apanese cedar (Cryptomeria japonica D. D o n ) p l an t ed

and d i s t r ibu ted in the K a n t o Dis t r ic t was extensive. They suggested a poss ib le l ink

to acid depos i t i on and pho tochemica l oxidants . Recent mon i to r i ng da t a indicate

tha t the average p H o f p rec ip i t a t ion in J a p a n ranges f rom 4.5 to 4.7, and tha t

p H values less than 4.0 are rare (CRIEPI , 1985; Tamak i and Hi rak i , 1986).

F o l i a r in jury and reduced g rowth have been d o c u m e n t e d in ac id ra in exper iments

at a p H of 3.0 and be low (Shr iner et al., 1974; W o o d and Bormann , 1974; J a c o b s o n

and Van Leuken , 1977; J a c o b s o n , 1980a, b; K o h n o and Fu j iwara , 1981, 1982).

However , ac id ra in exper iments on agr icu l tura l c rop species in J a p a n have not

been inves t iga ted to any extent.

This r epo r t descr ibes results o f an exposure exper iment on a selected cul t ivar

o f soybean us ing s imula ted ac id ra in to evaluate the g rowth effects o f ac id ra in levels found cur ren t ly in Japan .

Water, Air, and Soil Pollution 43:11-19, 1989. © 1989 Kluwer Academic Publishers. Printed in the Netherlands.

12 YOSHIHISA K O H N O A N D T A K U Y A KOBAYASHI

2. Materials and Methods

2.1. PLANT MATERIAL

Soybean seeds (Glycine max cv. Early Hakucho, Takii Seed Co., Kyoto, Japan) were sown in plastic pots (5 seeds to a pot, pot size: 11.3 cm diameter X 18 cm depth) filled with 1.5 L of an air dried volcanic ash soil. Young seedlings were thinned to one plant per pot 10 days after seeding. Solutions of mono-potassium phosphate and ammonium nitrate were applied to the soil prior to seeding to provide, N, P, and K at a rate of 200:200:200 kg ha -1.

A continuous rain generator system was used in this study (Figure 1; Kohno, 1987) to simulate acid rain. Plants were treated twice a week for a 1 hr period at a rate of 5.0 mm hr -1. Exposure treatments were begun 15 days after sowing and lasted for 7 weeks. The total amount of precipitation applied was 70 mm. Annual average precipitation at 80 sites in Japan is 1763 mm for the past 30 yr (Tokyo Astronomical Observatory, 1985). Since the amount of simulated rain during the experimental period was only one fifth of the weekly average, the plants were

supplementally irrigated with deionized water to avoid water deficit. Acid rain exposures were conducted in the early morning or after sunset to avoid

treatments under high temperatures and high irradiances during the day. The simulated rain naturally reached and penetrated surface soil in the pots. Pots were rerandomized prior to every exposure, to avoid shading effects. The experiment was conducted in a side-opened glasshouse and the plants were exposed to ambient

air. Plant leaves from each node were harvested separately and leaf areas were measured

with a leaf area meter after 1, 3, 5, and 7 weeks of exposure to acid rain. The leaves and stems were dried at 60 °C in a forced air oven and dry weights were

determined.

2.2. ACID RAIN SIMULATION

Acid rain solutions were prepared on the basis of precipitation chemistry data collected in Japan (Table I; CRIEPI, 1985). A stock solution was prepared containing sulfate, nitrate, and chloride anions in a concentration ratio of 2:1:1. This ratio of anions is similar to that observed and utilized in the United States (Jacobson et aL, 1980; NADP, 1986). Deionized water of pH 5.6 was used as the control and as a diluent for the stock solution. Measurements of rain pH were made with a digital pH meter (Model F-8AT, Horiba Ltd., Kyoto, Japan).

2.3. MEASUREMENT OF SOIL pH AND EC

Bulk samples of cultivated soils, taken before seeding and after harvesting plant tops, were dried at 60 °C in a forced air oven. A 20 g sample of dry soil was weighed into a plastic cup and 50 mL of deionized water was added. T h e s o i l suspension was stirred and allowed to stand for 30 min. Electric conductivity (EC)

E F F E C T S OF S I M U L A T E D ACID RAIN ON T H E G R O W T H OF SOYBEAN

" - " < l /

l

! " ~ I I I I I I I I I I I I I I I I I '

Storage tan ................

.... Droplets ......

• . • . ° • ° , . . • . . ° . ° ,

• . • • , . . . . . . o • . . • °

13

Needles

.__&

Gear motor Rain generator

K. ._

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l.am (L)

Gear mo to r

Fig. 1. Schematic diagram of rain generating system.

TABLE I

Characteristics of precipitation in the Inland Sea Region of Japan a,b

Parameter Average Range

pH Average 4.74 4.42 - 5.61 Lowest 4.08 3.71 - 4.54 Highest 5.66 4.87 - 6.69

EC (~S cm -a) 21.22

SO4 ~- (mg L l) 2.22

NO3 (mg L -l) 1.10

CI- (mg L- ' ) 0.98

a Period: April 1984. - June 1985. b Calculated from CRIEPI (1985). (N=24)

14 YOSHIHISA KOHNO AND TAKUYA KOBAYASHI

and pH of soil solutions were measured with a conductivity meter (Model DS- 8F, Horiba Ltd., Kyoto, Japan) and pH meter, in turn.

3. Results

3.1. VISIBLE INJURY

Acid rain treatment at pH 2.0 induced the formation of pale brown necrotic spots on the leaves within 24 hr after exposure. Necrotic symptoms first appeared along the veins and spread gradually into the interveinal areas. No injury symptoms were observed in young buds that developed prior to acid rain treatment. When leaves were exposed to acid rain at pH 2.0 during the early development stage, they became severely necrotic, crinkled and/or wrinkled. However, mature leaves were only slightly injured.

When young leaves were exposed to acid rain treatment at pH 3.0 during the

TABLE II

Leaf area of soybean plants as affected by simulated acid rain

Exposure Leaf position a period Rain (weeks) pH L-1 L-2 L-3 L-6/7

Leaf area (cm 2 plant -t)

1 5.6 40.5b b

4.0 39.6b 3.0 38.4b 2.0 28. la

3 5.6 75.5c 84.6b 4.0 72.2bc 77.3b 3.0 61.3b 76.0b 2.0 43.3a 53.4a

5 5.6 73.5c 81.9b 93.3b 37.8a 4.0 70.4bc 79.3b 96.4b 54.8a 3.0 59. lb 73.7b 89.2b 47.9a 2.0 38.8a 50.2a 50.5a 44.2a

7 5.6 73.3b 81.3b 96.6b 47.5a 4.0 71.7b 78.9b 97.9b 68. la 3.0 67.7b 78.8b 94.0b 48.6a 2.0 38.8a 55.7a 49.3a 55.9a

a Leaf position; L-1 to L-3: First (oldest) to third trifoliate leaf, L-6/7: Sixth or seventh trifoliate leaf.

b Within a column at each exposure period, any two means having a letter in common are not statistically different at the 5 % level by the Tukey's HSD multiple range test. (N=10)

EFFECTS OF SIMULATED ACID RAIN ON THE GROWTH OF SOYBEAN 15

early development stage, only slight injury was observed in the form of necrotic spots. However, mature leaves showed no visible symptoms of acid rain injury at pH 3.0 and greater.

Young fruits exposed to acid rain at pH 2.0 exhibited brown or dark brown necrotic spots on the surface of the pods. However, no injury symptoms were observed

on the pods at pH 3.0 or higher. No injury symptoms were observed in tissues of control plants (pH 5.6) or those exposed to acid rain at pH 4.0.

3.2. LEAF AREA

The leaf area of the first trifoliate leaf from soybean plants exposed to acid rain at pH 2.0 for 1 week was significantly smaller than that from the other treatments (Table II). After the 3rd and 5th weeks of treatment with acid rain at pH 3.0, the leaf area of first trifoliate leaf was significantly smaller than that for plants treated at pH 5.6. However, no difference was found in leaf area between pH 3.0 and 5.6 even after 7 weeks of exposure.

TABLE III

Tissue dry weights of soybean plants as affected by simulated acid rain

Exposure period Rain Whole (weeks) pH plant

Leaf position a

PL L-1 L-2 L-3 L-6/7 Others Stems Roots Pods

Dry weight (g plant -~)

0,32 010 0.16 0.06

5,6 0.62a b 0.16a 0.10b 0.21a 0.15a 4,0 0.63a 0.16a 0.09ab 0.22a 0.16a 3,0 0.62a 0.14a 0.09ab 0.23a 0.16a 2.0 0.62a 0.15a 0.08a 0.24a 0.15a

5,6 2.30b 0.19a 0.25c 0.27b 0.40b 0.66a 0.54bc 4.0 2.38b 0.19a 0.23bc 0.25ab 0.43b 0.69a 0.59c 3.0 2.09ab 0.19a 0.19ab 0.24ab 0.38ab 0.62a 0.47ab 2.0 1.88a 0.22a 0.17a 0.22a 0.31a 0.57a 0.38a

5.6 6.25b 0.24ab 0.26b 0.31a 0.41b 0.10a 1.65a 1.86b 1.21b 4.0 6.80b 0.24ab 0.25b 0.30a 0.42b 0.15a 1.83a 2.04b 1.37b 3,0 6.60b 0.19a 0.22ab 0.30a 0.39b 0.14a 1.90a 1.98b 1.22b 2.0 5.34a 0.26b 0.17a 0.24a 0.25a 0.16a 1.68a 1.57a 0.81a

5.6 10.91b 0.19a 0.25b 0.28b 0.40b 0.19a 2.18a 2.64b 1.65b 4.0 11.22b 0.18a 0.21b 0.27ab 0.38b 0.26a 2.19a 2.66b 1.84b 3.0 10.65b 0.15a 0.21b 0.29b 0.38b 0.19a 2.15a 2.53b 1.60b 2.0 8.72a 0.21a 0.14a 0.23a 0.22a 0.24a 1.87a 1.93a 1.00a

0.21a 0.24a 0.28a 0.21a

3.13a 3.23a 3.15a 2.88a

a Leaf position; PL: Primary leaves, L-1 to L-3: First (oldest) to third trifoliate leaves, L-6/7: Sixth or seventh trifoliate leaf, Others: Other younger and adventitious leaves.

b Within a column at each exposure period, any two means having a letter in common are not statistically different at the 5 % level by the Tukey's HSD multiple range test. (N=10)

16 YOSHIHISA KOHNO AND TAKUYA KOBAYASHI

At pH 2.0, the 2nd and 3rd trifoliate leaves were significantly smaller than those exposed to pH 3.0 or greater. But the 6th or 7th trifoliate leaves were not affected by acid rain treatment at any pH.

3.3 ~SSUE DRY WEIGHT

The total dry weight of plants exposed to acid rain at pH 2.0 for 5 and 7 weeks was less than that of plants exposed to acid rain at pH 3.0 or greater (Table III). The dry weights of first trifoliate leaves exposed to pH 2.0 for 1 week and those exposed to pH 3.0 for 3 weeks were less than those of leaves from control plants exposed to pH 5.6. Dry weights of stems and roots from plants exposed to acid rain at pH 2.0 for 5 and 7 weeks, were significantly less than those from other

pH treatments.

3.4 ROOT NODULES

Effective root nodules that were reddish brown or pink in the cross section (Allen and Allen, 1981), were counted after the exposure to acid rain for 3 and 5 weeks (Table IV).

After 3 weeks of acid rain treatment, the number of root nodules at pH 2.0 was significantly less than that at pH 5.6, otherwise there were no significant differences among any of the three acid rain treatments. After 5 weeks of exposure, the number of nodules at pH 3.0 was significantly less than that at pH 4.0 and that at pH 2.0 reduced to about half of that at pH 5.6.

3.5 SOIL pH AND EC

The initial pH of the cultivated soil prior to fertilization was about 6.0 and dropped to 5.4 after fertilizer application (Table V). After 3 weeks of acid rain treatments, there was no difference in soil pH among any of the four acid rain treatments. After 7 weeks of exposure, the pH of the soil in both control plants and acid

TABLE IV

Root nodules in soybean plants exposed to simulated acid rain

Exposure Rain pH period (weeks) 5.6 4.0 3.0 2.0

- - Nodules (number plant-~) a - -

3 5.0b b 4.lab 1.4ab 0.7a

5 20.5bc 27.2c 14.7ab 10.0a

a Number of reddish nodules in cross section. b Within a row, any two means having a letter in

common are not statistically different at the 5 % level by the Tukey's HSD multiple range test. (N=10)

EFFECTS OF SIMULATED ACID RAIN ON THE GROWTH OF SOYBEAN 17

TABLE V

The pH and EC of cultivated soils as affected by acid rain treatment

Soil sample pH a EC (txS cm-l) a

Initial 5.98 80.5 Fertilized 5.37 478.7

3-week exposure 5.6 5.47a b 287.5a 4.0 5.45a 261.0a 3.0 5.54a 312.2ab 2.0 5.39a 374.5b

7-week exposure 5.6 5.93c 97.7a 4.0 5.83b 94.2a 3.0 5.79b 113.1a 2.0 5.69a 159.8b

Soil: Deionized water=l:2.5. Within a column at each exposure period, any two means having a letter in common are not stati- stically different at the 5 % level by the Tukey's HSD multiple range test. (N=10)

rain treated plants tended to increase by about 0.3 to 0.4, thereby approaching

the pH of the initial unfertilized soil. The electric conductivity (EC) of unfertilized

soil was 80 txS cm -1 and increased to about 480 txS cm -1 after fertilizer application.

Conductivity of the soil at pH 2.0 after 7 weeks exposure was significantly greater

than that at any other pH.

4. Discussion

There have been many reports on the occurrence of visible injury induced by acid

rain treatment, since Shriner et al. (1974) first reported the findings of their acid

rain study. Jacobson (1980a, b) reported that the threshold for foliar injury

development was between pH 3 and pH 4 and Jacobson and Van Leuken (1977) found that continuous long-term exposure to acid rain at pH 3.4 caused injury

in sunflower (Hel ianthus annuus). Bean leaves exposed to acid rain from sulfuric

acid at pH 3.0 and below showed necrotic spots (Kohno and Fujiwara, 1981, 1982).

The threshold pH level for induction of visible injury in radish was between 3.3 and 4.0 (Johnston et al., 1986) and that for bush bean was between 3.2 and 4.0 (Johnston et al., 1982). In the current experiment, soybean plants exposed to the acid rain at pH 2.0 showed severe visible injury, but, little of any injury at pH 3.0. Therefore, the threshold value for induction of visible symptoms of acid rain

injury in soybeans appears to be approximately pH 3.0. Wood and Bormann (1977) observed that acid rain treatment at pH 2.3 stimulated

18 YOSHIHISA KOHNO AND T A K U Y A KOBAYASHI

plant growth in white pine (Pinus strobus) irrespective of leaf injury. Evans and Lewin (1980) reported that acid rain treatment at pH 3.1 and below decreased dry mass of seeds, leaves, and stems of pinto beans. Our results showed that acid rain treatment greatly reduced plant growth in soybean at pH 2.0, but had little or no effect on growth or dry matter accumulation at pH 3.0 and greater. These findings are consistent with field observations which indicate that the current pH level of ambient rain does not have an adverse effect on growth and yield of agricultural crops (EPRI, 1987; Fox et al., 1983; Irving, 1983).

In the present study, acid rain treatment at pH 2.0 caused a decrease in leaf area and leaf dry weight of soybean plants within a relatively short period of time after beginning exposure. Thus, characterizing the growth and appearance of young plants shortly after acid rain exposure may provide useful information in making preliminary assessments of the severity of acid rain injury in plants.

The slight stimulation in growth of plants at pH 4.0, in comparison to pH 5.6 controls may have been caused by an increase in absorption of nitrate-N through the leaf surface and soil during acid rain treatment. Evans et al. (1986), however, found that leaf absorption of N during acid precipitation was of no consequence, since the amount of N absorbed was less than that of the total N content in the leaves. Such stimulation in growth could be associated with an increased number of root nodules in plants exposed to acid rain treatment at pH 4.0. Therefore, changes in nutrient balance or recycling and in symbiotic soil microorganism activities of the surface soil layer should be considered in the assessment of acid precipitation effects on vegetation.

Lee et al. (1980) reported that even if visible injuries did not develop under simulated acid rain conditions, reduction in crop growth could be detected. Although soybean plants in the present study showed a reduction in both leaf enlargement and dry matter production at pH 2.0, only a single cultivar was studied. In order to generalize on the responses of agricultural crops to acid rain, several cultivars and species should be considered.

Acknowledgment

The authors extend their thanks to Dr. Donald T. Krizek, Plant Stress Laboratory, Natural Resources Institute, ARS, USDA, Beltsville, MD., U.S.A. for his critical reading of this manuscript and editorial suggestions.

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E F F E C T S O F S I M U L A T E D ACID RAIN ON T H E G R O W T H OF SOYBEAN 19

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