as calcium vanadate (Cannon, 1963; Hemphill, 1972; Lepp, 1977; Wallace et ..... of V in 30 shrubs (unidentified taxonomy) dispersely growing in areas in Idaho.
VANADIUM: TOXICITY AND A C C U M U L A T I O N BY BEANS
D. I. K A P L A N , D. C. A D R I A N O , * C. L. C A R L S O N , and K. S. S A J W A N
Biogeochemistry Division, Savannah River Ecology Laboratory, Drawer E, Aiken, SC 2980 1, U.S.A. (Received March 8, 1989; revised September 29, 1989) Abstract. Hydroponic and rhizotron studies were conducted to determine the effect of V on soybean [Glycine max (L.) Merr.] and bush bean (Phaseolus vulgaris L.) biomass and mineral nutrition. In the hydroponic study, 6 mg V L ~ (as VOSQ) in the nutrient solution drastically altered soybean nutrition, and significantly reduced plant biomass. Vanadium accumulated in the roots but not in the aerial portions of the plants. The data support the hypothesis that tissue V and Ca contents are related with high concentrations of both elements in the roots, and low concentrations in the aerial portions of the plant. Analysis of data with the Diagnostic and Recommendation Integrated System (DRIS) identified Ca as deficient in aerial tissues. The changes in Ca concentrations induced by V treatment may also have antagonized the concentrations of others macronutrients, most notably K and Mg. DRIS also indicated that K, Mg, and Zn levels were relatively high. The rhizotron study, which dealt with bush beans grown in metal-treated soils, further showed that V was primarily concentrated in the roots of the plants, with very little accumulated in the aerial portions.
1. Introduction
Vanadium is a trace metal which is ubiquitous in the natural environment, occurring as a constituent of many igneous rocks, shales, and uranium ores (Peterson and Girling, 1981; Adriano, 1986). During weathering of these materials, V is oxidized to vanadate (VO42-), which is soluble over a wide range of pH and is fairly mobile in soil (Peterson and Girling, 1981). Combustion of coal and oil for power generation is considered as the primary source of this metal in the environment (Adriano, 1986). While V is not considered an essential element for vascular plants, it is essential for certain bacteria and algae (Welch and Huffman, 1973). Vanadium can substitute for Mo in the nitrogenase enzyme required for nitrogen fixation by leguminous plants, so it can be beneficial to legumes (Amberger, 1975). Within the plant, it is believed that most V concentrates in roots, where it is thought to precipitate as calcium vanadate (Cannon, 1963; Hemphill, 1972; Lepp, 1977; Wallace et al., 1977; Peterson and Girling, 1981). Immobilization of V in this manner is presumed to be the primary mechanism of plant tolerance to large quantities of this metal (Peterson and Girling, 1981). Studies have demonstrated that plant species which absorb large amounts of Ca were also most tolerant to high V levels (Cannon, 1963; Peterson and Girling, 1981). Interactions of V within the plant have also been reported with Se (Cannon, 1963), Fe (Warington, 1954; Wallace et al., 1977; Basiouny, 1984), and Mn and Cu (Wallace et al., 1977). In a preliminary study conducted in this laboratory, it was noted that soybeans [Glycine m a x (L.) Merr.] grown in V-treated Hoagland's solution exhibited chlorosis * Author for all correspondence.
Water, Air, and Soil Pollution 49: 81-91, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.
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D. I. KAPLAN ET AL.
in the younger, upper leaves, but not in the older, lower leaves. Also, the roots of these stressed plants were darker in color, stubby, and produced fewer lateral branches. The objectives of this study were to further probe our initial observations by evaluating V accumulation in soybeans and bush beans, its effect on mineral nutrition, and tolerance of these species to this metal. 2. Materials and Methods
Both hydroponic and rhizotron studies were conducted to achieve these objectives. The hydroponic,study determined the mobility of V in soybean plants and its effects on macro- and micronutrient nutrition. The rhizotron study evaluated the uptake of V by bush beans (Phaseolusvulgaris L.). Results were then compared to determine if these studies conducted under differing conditions can be reconciled. 2.1. HYDROPONICSTUDY Soybean seeds were germinated in Perlite (tm), then transplanted at the first trueleaf stage to 2-L opaque polyethylene pots containing aerated, half-strength Hoagland's solution. The plants, 4 per pot, remained in untreated nutrient solution for seven days to acclimate from transplant shock. Concentrated vanadyl sulfate (VOSO4) solution was then added to obtain treatment levels of 0, 3, and 6 mg V L -1 (0, 0.6 and 1.2 mM V) in the nutrient solution. The plants had one trifoliate leaf when initially treated. The pots were arranged in a completely randomized design with each treatment replicated seven times. Plants were grown in a greenhouse under fluorescent lighting (16 hr days); day and night temperatures were 31 +_3 °C and 23 _+2 °C, respectively. Solution in pots were brought up to a 2-L volume daily with distilled water and adjusted to pH 5.5 _+0.1 twice weekly with 0.1 MHC1. The experiment was terminated 33 days after treatment by separating the plants into upper leaves (above middle node), lower leaves (below middle node), upper stem, lower stem, and roots. Similar plant parts from each of the four plants in a pot were composited and homogenized. The roots were carefully rinsed in three successive distilled-water baths. Plant tissues were oven dried at 55 °C for 10 days, weighed to the nearest 0.01 g, then ground to pass a 20 mesh (841 ~m) sieve. Ground samples were dried again overnight at 55 °C, after which 1.000 + 0.003 g of each sample was dry ashed at 500 °C for 4 hr and digested in HC1 following standard methods (Council on Soil Testing and Plant Analysis, 1980). The ashed samples were analyzed for Ca, Mg, K, and Fe by atomic absorption spectrometry. Nitrogen was determined using an Automatic Nitrogen/Carbon/ Sulfur Analyzer, P by the molybdenum-blue colorimetric method, and V by the gallic acid catalytic method of Weiguo (1983). Separate samples were digested using a nitric acid-perchloric acid solution and analyzed for B, Cu, Mn, Na, Mo, and Zn on an inductively coupled plasma emission spectrometer using standard methods (Council on Soil Testing and Plant Analysis,
VANADIUM~ TOXICITY AND ACCUMULATION BY BEANS
83
1980). For quality control, National Bureau of Standards tomato leaves (NBS, Washington, DC. NBS No. 1573) were carried through all analyses. Statistical analyses were performed using the SAS program. Interpretation of the tissue analysis was aided by using the Diagnosis and Recommendation Integrated System (DRIS) (Beaufils, 1973; Letzsch and Summer, 1983) DRIS is an interpretive technique used to determine which nutrient(s) are potentially limiting plant growth. It does this by comparing the ratio of nutrient concentrations of a plant to similar concentration ratios of healthy plants. 2.2.
RHIZOTRON
STUDY
Two 3 × 3 × 2 m rhizotron-lysimeter (rhizotron) cells were used in this study. The bottom of each cell was equipped with a drainage pipe and contained 10 cm of coarse sand (1.5 mmm) over 10 cm of pea gravel to facilitate water drainage. Orangeburg loamy sand (fine-loamy, siliceous, thermic Typic Paleudult) was placed, by horizon, in each cell and left for two years to attain densities similar to those found in the field. This soil had the following initial soil characteristics: pH of 5.5, 14.0 g O.M. kg -1, and a CEC of 2.73 cmol(+) kg -1. In the spring of 1987, the cells were thoroughly tilled, limed (2240 kg dolomitic limestone ha -1) and fertilized with (on a per ha basis) 67.2 kg N (as NH4NO3), 49 kg P (as P205), 93 kg K (as K20), 6.7 kg Mn (as MnSO4 HzO), 11.2 kg Zn (as ZnSO4 • 7H20), and 1.1 kg B (as H3BO3). There were two treatments, with metals (treated) and without (control). The treated cell received (on a per ha basis) 11.2 kg Cd (from CdC12), 5.6 kg Cr (from CrC13 • 6H20), 5.6 kg Ni (from NiC13 • 6H20), 5.6 kg T1 (from T12SO4), and 5.6 kg V (from VOSO4). Metals other than V were applied to the soil as part of a larger study which will not be reported here. The salts of these metals were dissolved in water, then evenly sprayed over the cells. The metals were immediately mixed into the top 15 cm of soil with a rototiller. The control cell did not receive any metals. In the spring of 1988, bush beans were planted with a final spacing of 60 cm between rows and 10 cm between plants within a row. There were five rows per plot, of which only the middle 1.8 m of the three central rows were harvested. Irrigation was provided when needed. Each row was harvested separately and treated as a subplot. Mature bean pods were harvested four times during the growing season while the upper leaves (second from top), lower leaves (first trifoliate), and roots were collected during the third harvest for chemical analysis. Roots were thoroughly washed by carefully rinsing in four successive tap-water baths followed by a final rinsing in a distilled water bath. Plant tissue was dried, weighed, then chemically analyzed as described above.
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3. Results and Discussion 3.1. HYDROPONICSTUDY
Visual Symptoms Both the 3 and 6 mg V L -1 treatments induced visual toxicity symptoms, with severity increasing with the V level. Eight days after treatment, the roots of the higher V-treated plants started to darken, followed four days later by a reduction of lateral root formation. At this time, the roots displayed a 'clubbing' appearance, i.e., the lateral roots became shorter and thicker and the tips were 'bulbous', as opposed to tapered. At about the time that roots started to darken, the leaves in the upper canopy started to manifest chlorosis. Ten days later, the chlorosis had spread to the lower leaves. Plants from both treatments exhibited necrosis only in the first monofoliate leaves. Towards the end of the experiment, some reddening occurred in the petioles and stems. Warington (1956) reported similar reddening in stem tissue, as well as in the leaf tips, of sorghum (Sorghum vulgare L.) plants grown in hydroponic solutions spiked with V.
Plant Biomass Biomass estimates for the 3 mg V L -1 treatment generally did not differ statistically from the control, while all biomass estimates for the 6 mg V L -~ treatment were significantly ( P < 0 . 0 5 ) lower than those for the control (Table I). Upper stems, upper leaves, lower stems and lower leaves of the high V-treatment plants had similar reductions in biomass, i.e., 40 _+ 2% less than the control. Root weights were the least adversely affected, showing a 22% decrease with respect to the control. However, a striking change in root morphology occurred in the V-stressed plants. While the roots of affected plants grew shorter and produced fewer branches, they also thickened, resulting in a greater weight per unit length. Vanadium concentration in nutrient solution was significantly and inversely correlated to all biomass estimates, including total leaf weight (r = -0.78, P < 0.001) and root weight (r = -0.45, P < 0.05). TABLE I Weights of soybean tissues grown in Hoagtand's solution treated with varying levels of V (as VOSO4). V conc.
Total plant
Total leaf
Total stem
Upper leaf
Mg L-l 0 3.0 6.0
L o w e r Upper leaf stem
Lower stem
Root
3.72a 3.15a 2.26b
2.99a 2.30b 1.92c
2.07a 2.12a 1.62b
g 15.40aa 13.41a 9.62b
8.40a 7.22a 4.91b
4.94a 4.12b 3.09c
4.68a 4.07a 2.66b
1.95a 1.82a 1.16b
a Means in a column followed by the same letter are not significantly different (P
