Experimental approach to aluminum phytotoxicity

MICROCHEMICAL

JOURNAL

Experimental

46,

20-29 (1992)

Approach

to Aluminum

Phytotoxicity’

S. COSTANTINI, *J R. GIORDANO,* E. BECCALONI,* C. PERANI,~’ AND S. GREGO~ *Istituto Superiore di Sanitri Viale Regina Elena, 299 00161 Rome, Italy; and tUniversity “La Tuscia” Department of Agrobiology and Agrochemistry Via S. Camillo de Lellis, Blocco B 01100 Viterbo, Italy Received August 12, 1991; accepted December 14, 1991 Soil acidity is a serious agricultural problem throughout large geographic areas of the world, possibly affecting as much as 40% of the world’s arable soils. Plants growing on acid soils suffer from a number of detrimental factors, including nutrient deficiencies, drought intolerance, and Mn toxicity. Aluminum toxicity, however, has been identified as one of the more important growth-limiting factors on acid soils. The intense research effort addressing basic and applied aspects of Al phytotoxicity reflects the agronomic and economic importance of the acid soil-Al problem. In the first stage of this study, several 21-day-old lettuce plants (Lactuca sativa L.) were transplanted from normal soil (pH 6.8, exchangeable Al 0.11 meq/lOO g soil) to an acid soil (pH 3.8, exchangeable Al 10 meq/lOO g soil). Following that, the lettuce plants were collected at random at different times over a period of 14 days and the Al, Ca, and K contents were determined in both roots and leaves by means of atomic absorption spectrometry. Results indicated that plants grown in the acid soil accumulate considerable amounts of Al, especially in the roots, where the loss of cations was more evident. In comparison with the “blank plants,” a reduced growth was also noted. In a second stage of the study, the lettuce plants from normal soil were allowed to grow in nutrient solutions having different Al concentrations, namely 2, 10, and 50 nuW. Sample collection was performed after 2,4,8,24, and 48 h, respectively. Aluminum, Ca, K, and Mg were determined as described above. In roots, high accumulation factors of aluminum were quickly obtained but the decrease of Ca seemed to be lower than that observed in the first model. In the leaves, the intake of Al was less and the changes in cation concentrations were limited and constant in all solutions. 8 1992 Academic Press, Inc.

INTRODUCTION

Aluminum is the most abundant metal and the third most common element in the earth’s crust, where it is locked in soils and minerals as oxides, or more commonly, as complex aluminosilicates. Despite its abundance, Al is present in very small amounts in living organisms and it is usually excluded from normal biochemical and metabolic processes because of its chemical nature. Owing to its high electric charge and small radius (0.51 A), the element is too reactive to be found free in nature. The only accessible oxidation state in biological systems is + 3, but A13+ has never been demonstrated as having a definite biological funci Submitted in conjunction with the Fifth Italo-Hungarian Symposium on Spectrochemistry: ity Control and Assurance in Life Sciences, Pisa, Italy, September 9-13, 1991. 2 To whom correspondence should be addressed. 20 0026-265x/92 $4.00 Copyright Au rights

0 1992 by Academic Press, Inc. of reproduction in any form reserved.

Qual-

ALUMINUM

PHYTOTOXICITY

21

tion; this suggests that the ion does not possess properties which are compatible with fundamental processes (14). In aqueous solutions, the ion protolyzes part of the water envelope and forms hydroxo complexes; as a result, the solution becomes acidic. At neutral pH, Al minerals are extremely insoluble and the concentration of dissolved A13+ is therefore low in both surface and subsoil waters. Nevertheless, the acidification caused by acid rain and the wide use of acidifying fertilizers obviously increase the concentration of soluble Al in soil and rivers (I). Concentration levels in natural fresh waters vary from a few tens to some hundreds of micrograms per liter, whereas the concentration of Al dissolved in seawater is generally lower than 1 p&liter (2). In the agronomical field the element plays an important role as a material used by the industry in preparing and transforming foods; for this reason, the potential risk for consumers must be considered. Even though toxic effects of the element have primarily been demonstrated within the ambit of dialysis, numerous findings indicate that Al may also exert its action in people who do not have any renal damage. In fact, the element seems to be implicated in the pathogenesis of Alzheimer’s disease, even if its role has not yet been explained by current evidence (5-n. In plants Al toxicity seems to act at the growth level and its accumulation is a function of the acidity of the soil which influences its bioavailability. Since soil acidity is a serious agricultural problem throughout large areas of the world, Al toxicity represents one of the more important growth-limiting factors on acid soils. Plant tolerance to Al seems to be due to the combination of exclusion mechanisms and internal tolerance, through the formation of a pH barrier induced in the rhizosphere, and also the action of radical exudates which have chelating properties (8) I In the present study we experimented with two models of growth and Al absorption in lettuce plants (Lactuca sativa L.), in both normal and acid soils, and also in nutrient solution. The determination of Al, Ca, and K was performed in both models, whereas the determination of Mg was made only in the second study. Either flame or flameless atomic absorption spectrometry (AAS) techniques were employed in all measurements. MATERIALS

Experimental

AND METHODS

Models

In the first model, small lettuce plants 3 weeks old were transplanted to an acid soil which had the following characteristics: potential acidity, pH 3.4; real acidity, pH 3.8; exchangeable Al, 10 meq/lOO g (33 mmoYkg). As control, lettuce plants that were grown in normal soil (pH 6.8; exchangeable Al, 0.11 meq/lOO g) were used. The lettuce plants were randomly collected at different times (0, 2, 6, 12, and 14 days) and the determination of the elements was performed in both roots and leaves. In the second model, the lettuce plants were allowed to bud in a Hogland-type nutrient solution which had the following composition (in mg/liter):

22

COSTANTINI

CaW03)2 KN03 Mg (NO,), K,HPO, Fe-EDTA H3BO3

M&O, ZnSO, cuso, Na,Mo

* 2H,O

ET

AL.

236.0 75.6 83.2 0.070 7.32 0.49 0.034 0.056 0.048 0.34

After 3 weeks, the plants were randomly transferred to a fresh nutrient solution also containing Al at concentrations of 2, 10, and 50 mM, respectively. The samples were collected after 0, 2, 4, 8, 24, and 48 h from the beginning of the experiment. A group of plants, placed in hydroponics, which was not spiked with Al and which had the same acidity of the test solutions, was used as a control. Also in this study, the element concentrations were measured in both roots and leaves. Sample Treatment

The lettuce plants from each treatment were analyzed in pools of 10 individuals. After an accurate washing with doubly distilled water, sub samples of roots and leaves were dried at 105 + 3°C in order to determine the water content. About 0.1 g of dried material reduced to a fine powder was weighed in a quartz crucible and then dry-ashed at 450°C for 24 h. After addition of 1 ml of water and 200 pl of concentrated HNO, (Suprapur Merck, Germany), the solution was evaporated to dryness on a hot plate at 120°C; after that, the sample was placed again in the electric muffle at 450°C. The whole procedure was repeated until white ash was obtained. The residue was dissolved in 1 ml of concentrate HNO, and 500 pl of concentrated HCl. Finally, the volume of the solution was brought up to 25 ml with water. All samples were analyzed in triplicate and the results averaged. To avoid contamination problems from exogenous Al, all the necessary precautions were adopted. Chemical

Analysis

The determinations of Ca, K, and Mg were performed by the air-acetylene flame AAS technique, after dilution of samples with a La solution (0.2% for Ca, 0.1% for K, and 0.1% for Mg). A Perkin-Elmer 5100 spectrophotometer with a continuum source background correction system was used. In relation to its concentration in samples, the Al determination was performed by either the nitrousacetylene flame or flameless AAS techniques. The latter method was adopted when the Al concentration in the analyzed solution was lower than 5 mg/liter. A Varian 300 zeeman spectrophotometer, equipped with a GTA 96 Z graphite furnace and a PSD 96 autosampler was employed in the flameless mode; pyrolytic tubes with 1’Vov platforms were used in the furnace. The sample was diluted by a 2.5 g/liter solution of Mg(NO,)* used as a matrix modifier. Calibration was done

ALUMINUM

23

PHYTOTOXICITY

by the standard additions method. In all measurements, the sample dilution ratio was determined in order to remain within the linear range of analytical response. The furnace program adopted in the flameless evaluation of Al is reported in Table 1. RESULTS AND DISCUSSION

General results are reported in Tables 2 and 3, whereas Table 4 shows data on the quality control tests for Al. The variations in the Al/cation ratio vs the growth times are shown in Figs. l-9. In the first model, the roots of plants grown in acid soil, in which the Al bioavailability was 100 times higher than that in the normal soil, showed a tendency to absorb heavy amounts of the element. After 2 weeks of growth in the acid environment, the accumulation factor in roots was 3.4 relative to that of the control. Both Ca and K concentrations considerably decreased as the amount of Al absorbed increased. The Ca concentration changed from 1.22 to 0.16 g/100 g dry weight (dw), with a loss factor of 7.6 (86.8%). The decrease of K seemed to be less dramatic, changing from a concentration of 3.62 to 2.26 g/100 g dw, with a loss factor of 1.6 (37.5%). In the roots of samples from normal soil the concentration changes of all elements studied were not statistically significant and formed part of the biological variability of the model adopted. In leaves, the highest Al concentration found after 14 days of growth at 3.8 pH (318 mg/kg dw) was considerably lower than that obtained in roots (2880 mg/kg dw). The decrease of Ca appeared less evident with a loss factor of 1.9 (47.9%), whereas the K loss was comparable to the one observed in the roots. After only 2 h from the beginning of the second experiment, the Al concentration found in the roots of plants grown in the nutrient solution containing 2 mil4 Al (2848 mg/kg dw) was comparable to the maximum level obtained after 2 weeks of growth in acid soil. In general, the concentration levels of the element quickly rose along with the concentration of Al in the solution. In roots of samples from nutrient solutions containing 2 and 10 mA4 Al, the decrease in Ca seemed to be lower (loss factor 2.3, i.e., 56.9%, and 3.3, i.e., 69.9%, respectively) than that observed in the first model; in contrast, the results obtained using an Al concentration of 50 mM were comparable to those found in soil. The K level also decreased in relation to the Al concentration in the nutrient TABLE 1 Aluminum Flameless AAS Determination: Step Drying I Drying II Ashing I Ashing II Atomizing Cleaning Cooling

Temperature (“Cl 90 130 700 1200 2600 2700 40

Furnace Parameters

Ramp time w

Hold time w

Gas flow (literslmin)

10 10 20 20 0.7 1 13

10 10 15 10 5 5 10

3.0 3.0 3.0 3.0 0 3.0 3.0

Note. Tube: pyrocoated with 1’Vov platform. Gas flow: Argon UPP. Sample volume: 10 ~1.

24

COSTANTINI

Al, Ca, and K Concentrations

ET AL.

TABLE 2 in Roots

and Leaves

(Model

pH 6.8

1) pH 3.8

D”

Al

Ca

K

Al

Ca

K

0 2 6 10 12 14

848 825 790 870 972 828

1.22 1.18 0.98 0.87 1.08 0.92

3.62 4.12 3.95 3.22 3.23 3.45

848 1550 1590 1970 2920

1.22 0.31 0.21 0.16 0.17 0.16

3.62 3.10 2.12 2.31 2.33 2.26

0 2 6 10 12 14

107 125 157 141 134 160

1.44 1.78 1.74 1.56 1.68 1.77

Leaves 3.56 3.12 3.92 3.52 3.14 3.20

107 266 306 336 315 318

1.44 1.04 O.% 0.92 0.77 0.75

3.56 3.06 2.78 3.05 2.62 2.44

Note. Al, mg/kg dw; Ca, K, g/100 g dw. a D, days of growth.

TABLE Al, Ca, K, and Mg Concentrations

3 in Roots

and Leaves

(Model

Roots

2) Leaves

C”

T

Al

Ca

K

Mis

Al

Ca

K

Mg

0 0 0 0 0 0 2 2 2 2 2 10 10 10 10 10 50 50 50 50 50

0 2 4 8 24 48 2 4 8 24 48 2 4 8 24 48 2 4 8 24 48

623 684 646 674 666 693 2848 3130 3012 4900 5250 3240 3570 4690 6590 7150 4840

1.23 1.12 1.18 1.35 1.11 1.39 0.90 1.02 0.68 0.57 0.53 0.53 0.48 0.50 0.53 0.37 0.26 0.17 0.17 0.19 0.18

3.98 4.26 3.86 4.48 3.70 4.08 3.80 3.64 3.42 3.12 3.16 3.18 2.% 2.58 1.96 1.98 3.28 2.48 1.64 1.48 1.56

0.22 0.19 0.24 2.30 2.11 2.42 0.24 0.23 0.21 0.19 0.18 0.22 0.24 0.18 0.16 0.18 0.21 0.16 0.18 0.11 0.13

106 112 114 109 116 121 109 102 94 127 144 98 126 148 152 204 214 277 328 395 416

1.94 1.86 1.98 1.98 1.90 2.08 1.84 1.69 1.76 1.66 1.45 1.83 1.72 1.65 1.68 1.52 1.78 1.69 1.65 1.68 1.51

3.68 3.26 3.42 3.89 4.00 3.90 3.66 3.36 3.12 3.08 2.96 3.08 2.66 2.88 2.16 1.56 2.86 2.36 2.48 1.28 1.14

0.39 0.32 0.38 0.40 0.41 0.39 0.38 0.34 0.35 0.33 0.34 0.39 0.42 0.36 0.34 0.35 0.35 0.34 0.38 0.37 0.34

6260 6980 7850

Note. Al, mgkg dw; Ca, K, Mg, g/l00 g dw. 0 C, aluminum concentration (mbf) in nutrient

solution.

T, time (h) of growth.

ALUMINUM

25

PHYTOTOXICITY

TABLE 4 Aluminum Data of Quality Control Tests Recovery Flame AAS method Flameless AAS method

107% %% Precision Coefficient of variation (%o) 6.7 5.1 3.8

Whole analytical procedure Flame measurements Graphite furnace measurements Detection limit (graphite furnace) Measurements Whole procedure

3 &liter 1000 &liter

solution. The loss factor was 1.3 (20.6%) in 2 mM Al, 2.0 (50.2%) in 10 m&f Al, and 2.6 (60.8%) in 50 nut4 Al, respectively. The loss of Mg was slight and constant at the different Al concentrations. In the leaves, the Al intake was less and the highest level found in samples from 50 nut4 Al solution (416 mg/kg dw) was lower than the baseline value found in roots (623 mg/kg dw). The changes in Ca concentrations were limited and constant in all solutions. Magnesium also did not present significant changes but K had a loss factor higher than those found in the roots. Many authors have suggested that Al induces mineral deficiencies and that these deficiencies form the basis of Al phytotoxicity. The toxic effect probably depends on the plant species and the extent of Al stress. The most evident effects

G & v25

17 16 15 14 1312lllo-

-

0

2

Days FIG.

growth

4

of

ii

10

12

soil

1. Growth of lettuce plants in soil: AVCa ratio in roots.

14

26

COSTANTINI 1.5

ET AL.

,

1.4

-

1.3 1.2

-

1.1

-

F A r x

l0.9

-

0.6

-

.p

0.7

-

E

0.6-

5

0.5

-

a

0.4

-

0.3

-

0.2

-

0.1

-

04,

,

0

,

2

,

,

, irowth

4

Days

,

,

,

,

in8

of

,

,

10

,

12

14

soil

FIG. 2. Growth of lettuce plants in soil: Al/K ratio in roots.

observed in our first study under conditions of Al stress were a reduction in both root and shoot growth, accompanied by a late maturation of leaves, a loss of the chlorophyll content, and necrosis of leaf tips. In particular, the elongation of the main axis of the roots was inhibited and the roots became thickened, brittle, and necrotic. The growth reduction of roots ranged from 15 to 35% of controls, whereas the reduction of the shoot growth was heavier (75%). Previous studies (WZ) have suggested that Al could interfere with the absorption and transport of Ca in plants. Our observation that Ca concentrations decreased in both roots and shoots of L. sativa under conditions of Al stress agrees with that of other authors who have previously observed this phenomenon in other species of plants. Reduced concentrations of Mg in plants caused by Al have also been reported; however, it seems likely that Al-induced Mg deficiency does 4.5 4

ph 3.8 - AVCa

i;i 3.5 A 3 25 $ 2.5 E E

2

s

m 1.5 u a

ph 3.8 : AI/K

1

pH 8.8 - AI/Ca

0.5 0

ph 6.8 - AI/K

I 0

2

4

Days

6

of growth

a

10

12

14

in soil

FIG. 3. Growth of lettuce plants in soil: AUCa or Al/K in leaves.

ALUMINUM

27

PHYTOTOXICITY

45 40

2mMAl

,

Control 0

,

40

Hours

of gzwth

in hydroponics

FIG. 4. Growth of lettuce plants in hydroponics solution: Al/Ca ratio in roots. 2.6 2.6

-

2.4

-

2.2

,

10mM

0 FIG.

Hours

in hydroponics

4o

5. Growth of lettuce plants in hydroponics solution: AYCa ratio in leaves.

0

20

Hours FIG.

of gr%‘wth

of growth

40

in hydroponics

6. Growth of lettuce plants in hydroponics solution: Al/K ratio in roots.

28

COSTANTINI

s & 7

3.6 3.4 3.2 3 2.6 2.6 2.4 2.2

-5 0 z m ;

1.6 1.6 1.4

a

ET AL.

2

1.21 0.6 0.6 0.4 0.2 40

Hours

of &wth

in hydroponics

FIG. 7. Growth of lettuce plants in hydroponics solution: Al/K ratio in leaves. 70 60

, -

v-

2mMAl

+

Control 0 I 0

40

20

Hours

of growth

in hydroponics

FIG. 8. Growth of lettuce plants in hydroponics solution: AlLMg ratio in roots.

1OmMAl

40

20

Hours

of growth

.

in hydroponics

FIG. 9. Growth of lettuce plants in hydroponics solution: Al&Q ratio in leaves.

ALUMINUM

PHYTOTOXICITY

29

not represent a direct injury by Al, but is part of the total Al toxicity syndrome. Our results obtained in the second study show that in lettuce plants the loss of Mg is very limited; this observation disagrees with the one reported for Zea mays (8) where, under conditions of Al stress, the levels of Mg in leaves and roots decreased more than those of other cations. The considerations made for Mg are also valid for K. In conclusion, the results obtained in both models indicate the existence of a correlation between the amounts of Al absorbed and the loss of cations. This phenomenon could be a result of either an inhibiting effect of the Al or a partial loss of the cations caused by the acidity of the environment. REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

Martin, B. R. Bioinorganic chemistry of aluminum. In Metal Ions in Biological Systems (P. Siege1 and A. Siegel, Eds.) Vol. 24, pp. 1-57. Dekker, New YorWBasel, 1988. Driscoll, C. T.; Schecher, W. D. Aluminum in the environment. In Metal Ions in Biologica/ Systems (P. Siege1 and A. Siegel, Eds.) Vol. 24, pp. 59-122. Dekker, New YorWBasel, 1988. Nyholm, N. E. I. Environ. Res., 1981, 26, 363-371. Katzman, R. N. Engl. J. Med., 1986, 314, 964-973. Alfrey, A. C.; LeGendre, G. R., Kaehny, W. D. N. Eng/. J. Med., 1976, 294, 184-188. Dunea, G.; Mahurkar, S. D.; Mamdani, B.; Smith, E. C. Ann. Znt. Med., 1978, 88, 502-W. Glenner, G. G.; Wong, C. W. Biochem. Biophys. Res. Commun. 1984, 122, 1131-1135. Taylor, G. J. The physiology of aluminum phytotoxicity. In Metal Ions in Biological Systems (P. Siegel and A. Siegel, Eds.) Vol. 24, pp. 123-163. Dekker, New YorWBasel, 1988. Foy, C. D.; Fleming, A. L.; Armiger, W. H. Agron. J., 1969, 61, 505-511. Soileau, J. M.; Engelstad, 0. P.; Martin Jr., J. B. Soil Sci. Sot. Am. hoc., 1969, 33, 919-924. Edwards, J. H.; Horton, B. D.; Kirkpatrick, H. C. J. Am. Sot. Hort. Sci., 1976, 101, 139-142.