The influence of arsenite concentration on arsenic accumulation in tomato and bean plants

SCIENTIA HORTICULTuRR ELSEVIER

Scientia Horticulturae 71 (1997) 167-176

The influence of arsenite concentration on arsenic accumulation in tomato and bean plants A.A. Carbonell-Barrachina a,* J, F. Burl6 a, A. Burgos-Hernkdez E. L6pez a, J. Mataix a

b,

a Departamento de Agroquimica y Bioquimica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, Alicante 03080, Spain b Food Science Department, Louisiana State University, Baton Rouge, LA 70803.4200, USA

Accepted 11 August 1997

Abstract by tomato (Lycopersicum esculentum Mill) and bean (Phaseolus in nutrient solution was examined. The processes of As uptake and accumulation among roots, stems, leaves, and fruit were studied. Tomato and bean plants were grown in nutrient solution containing four levels of arsenite: 0, 2, 5, and 10 mg As l- ’ . Arsenite was phytotoxic to both plant species; tomato plants, however, were more tolerant to As pollution than bean plants. Bean plants exhibited symptoms of As toxicity, and plants treated with 10 mg As I-’ were dead after 36 days of treatment. In tomato, As exposure resulted in a significant reduction in dry biomass production but tissue chlorosis or necrosis were not observed. The strategy developed by tomato plants to tolerate As was avoidance; limiting As transport to shoots and increasing As accumulation in the root system. Arsenic in tomato root tissue seemed to be so effectively compartmentalized that its impact in plant growth and metabolism was minimal. However, in bean plants upon uptake, As was readily transported to shoots and accumulated to high concentrations in leaf tissue. The observed differential absorption and translocation of arsenite or its metabolized species by tomato and bean plants were probably responsible for the different plant tolerance to As pollution. 0 1997 Elsevier Science B.V. Arsenic

(As) absorption

vulgar-is L.) as affected by arsenite concentration

Keywords: Arsenic pollution; Lycopersicum esculentum; Phaseolus vulgaris; Phytotoxicity

* Corresponding author. Tel.: + 1 504 388 6422; fax: + 1 504 388 6423; e-mail: [email protected] ’ Present address: Wetland Biogecchemistry Institute, Louisiana State University, Baton Rouge, LA 70803-75 11. USA. 0304-4238/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO304-4238(97)00114-3

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1. Introduction High levels of arsenic (As) are present in some agricultural soils because arsenicals (As compounds) have been used extensively as pesticides, herbicides, and fungicides. Arsenic can be toxic to plants and may accumulate in plants and thereby enter the animal and human food chain. The total amount of As in soil and its chemical forms have an important influence on plant growth and animal and human health (Yan-Chu, 1994). Sodium arsenite (NaAsO,) is used as a pesticide in vineyards as treatment for Stereum hirsutum, Phomopsis viticola, Sparganothis pilleriana, and Pseudococcus citri (Carbonell-Barrachina et al., 1994; Carbonell-Barrachina et al., 1995a, Carbonell-Barrachina et al., 1995b). Arsenite is carefully applied (as a spray) in a winter treatment to the stocks while they are dormant (MAPA, 1988). Careless application of pesticide often results in drift to non-target crops (Wauchope, 1983). Due to the historical use of inorganic arsenicals (such as sodium arsenite) in agriculture, there is a legacy of contaminated orchard soils. In Spain, soils where sodium arsenite was widely applied are now frequently used for tomato and bean growing (Carbonell-Barrachina et al., 1994; Carbonell-Barrachina et al., 1995a, Carbonell-Barrachina et al., 1995b). Arsenate [As(V)] and arsenite [As(III)] are the primary As forms in soils (Masscheleyn et al., 1991a). Soil As can undergo a variety of reactions: oxidation-reduction (Brannon and Patrick, 1987; Masscheleyn et al., 1991a) and methylation-demethylation (Brannon and Patrick, 1987). Methylated arsenic oxyacids can be produced by a variety of microorganisms, and their presence has been reported in a wide range of soils, sediments and waters (Masscheleyn et al., 1991a). At high soil redox levels (500-200 mV>, typical Eh of the mineral soils where tomato and bean plants grow, the major part (60-90%) of the As is present as As(V) (Masscheleyn et al., 1991a). However, chemical kinetics play an important role in the conversion between As(V) and As(II1). In this way, although thermodynamically unstable, considerable amounts of As(V) and As(II1) were found under highly reduced and oxidized conditions, respectively (Masscheleyn et al., 1991a). The present paper deals with a situation, in which the major part of the As applied as arsenite remained as As(II1) (the most phytotoxic specie, Marin et al., 1992). Arsenic absorption by plants is influenced by many factors including plant species (Walsh and Keeney, 1975), the concentration of As in the soil (NAS, 1977), soil properties such as pH and clay content (Von Endt et al., 1968), and the presence of other ions (Khattak et al., 1991). Reported As content of plants grown on soils with no prior arsenic-containing pesticides application varied from 0.01 to about 5 mg As kg-’ dry weight (NAS, 1977). Plants grown on arsenic-contaminated soils contained high As levels, especially in the roots (Wauchope and McWhorter, 1977; Carbonell-Barrachina, 1992; Carbonell-Barrachina, 1995). The largest quantities of plant As residues are found in roots, medium amounts of residues are found in the above ground vegetative parts (leaves and stems), and the smallest contents are found in fruit and seeds (Walsh and Keeney, 1975; Carbonell-Barrachina, 1992; Carbonell-Barrachina, 1995). As (III) has a high toxicity for radicular membranes (Sachs and Michaels, 1971),

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because As reacts with sulthydryl groups of proteins @peer, 1973) causing disruption of root functions (Orwick et al., 1976) and even cellular death. The fact that some plants may accumulate As concentrations greater than 0.1% of their dry weight (levels which would kill other plants) dramatically illustrates that plant species differ in their tolerance, if not their uptake capability, for As. Reported ranking of relative sensitivity to As are (in order of increasing sensitivity; Wauchope, 1983): asparagus, tomato, potato, carrot, tobacco, grape, raspberry < strawberry, sweet corn, beet, squash < bean, onion, pea, cucumber and alfalfa. Since bean and tomato plants differ in their sensitivity to As, the main objective of this paper was to determine whether these plants have different strategies for preventing As toxicity, and whether the different strategies influence As uptake, distribution, and accumulation in plants. The effects of arsenic, applied as sodium arsenite at different rates, on the growth of tomato and bean plants and the distribution of the absorbed As among roots, stems, leaves, and fruit are reported.

2. Materials

and methods

Tomato plants (Lycopersicum esculentum Mill) cultivar Marmande and bean plants (Phaseolus vulgaris L.) cultivar Buenos Aires were cultivated under greenhouse conditions, using crushed volcanic rock as inert media for cultivation. The treatments consisted of one chemical form of As [A&II), applied as sodium arsenite, NaAsO,] with four concentrations: 0, 2, 5, and 10 mg As(II1) 1-l. Treatments were replicated 4 times. The As in solution was analyzed regularly using a hydride generation technique with cold trapping and atomic absorption spectrophotometry (Masscheleyn et al., 1991b) to verify that the chemical form of the added As did not change over time. Arsenite was found to be stable with respect to oxidation-reduction and methylation-demethylation reactions for a period of 4 days. Thus, the nutrient solution was replaced every 4 days in order to maintain the desired treatments. Seeds were germinated in sterilized volcanic rock. Fourteen days after germination, uniform seedlings were selected. The volcanic rock was washed from the root system with distilled deionized water and seedlings transferred to hydroponic pots containing 1 1 of nutrient solution. A single pot, representing a specific As rate treatment, contained one seedling. The basal nutrient solution (Feigin et al., 1987) contained (in mg 1-l): 126 N; 46.5 P; 136.9 K; 31.6 Mg; 160.5 Ca; 2.0 Fe; 0.8 Mn; 0.5 B; 0.3 MO; and 0.2 of Zn and Cu. After 14 days of acclimatization to the hydroponic culture, plants were grown for 36 days, and then harvested (plants were 64-day old after germination). Tomato and bean plants were grown in greenhouse conditions during the months of late April, May and June; this season can be considered as being in between the early and the normal cycles for these two annual plants under greenhouse conditions in Alicante (Eastern Spain). The developmental stage of the tomato and bean plants at the harvest time was maturity of the first fruit set. Tomato and bean plants started their fruiting period after 14 d of treatment (plants were 42-day old after germination). Due to the small sample size

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obtained for several treatments, all fruit (mature and non-mature) were combined and analyzed together. This stage of development was selected as the sampling period since in a previous study (Carbonell-Barrachina et al., 19971, bean plant growth was dramatically affected by arsenite applications at rates of 2.0 and 5.0 mg As l- ‘, and plants died after approximately 60 days of treatment (ripening of the second fruit set>. Roots were washed with tap water and rinsed several times with distilled-deionized water. Roots, stems plus branches, leaves, and fruit were separated and yields were determined on a wet weight and dry weight (60-70°C for 72 h) basis. Samples were ground in a stainless-steel mill to obtain a homogeneous sample. Plant tissue samples were digested using a wet ashing technique, digesting 0.5 g sample in 5 ml concentrated HNO,. Temperature during digestion was controlled to a maximum of 120-130°C to avoid As volatilization (Marin et al., 1993). Digested samples were filtered and diluted with distilled-deionized water to 35 ml. Arsenic in the extracts was determined with a with a Perkin Elmer Optima, Model 3000, Inductively Coupled Argon Plasma Emission Spectrometer (ICP). Acid blanks were analyzed in order to assess possible As contamination. The As content of the HNO, used was below the detection limit of the ICP (40 pg 1-l). Statistical analyses were performed using procedures available in SAS @AS, 1987).

the PROC

ANOVA

and PROC

GLM

3. Results and discussion 3.1. Plant growth Tomato and bean plant growth (root, stem plus branches, leaves and fruit dry matter production) were significantly affected by As treatments. Increasing levels of A&II) in the nutrient solution significantly decreased tomato and bean plant dry matter production (Table 1). However, tomato biomass production was less affected than bean dry matter production by the arsenite treatments. A steady decrease in root, stem and leaf dry biomass productions of tomato and bean plants were observed with increased A&II) concentrations in the nutrient solution (Table 1). Root plus shoot dry biomass production of tomato plants was reduced to 87.2, 50.2 and 42.2% as compared to controls by the As treatments 2, 5, and 10 mg As l-‘, respectively. This growth parameter was more severely restricted in bean plants and it was reduced to 38.1 and 26.3% as compared to controls by treatments of 2 and 5 mg As l- ‘, respectively. In the same way, tomato and bean fruit productions were significantly reduced by increasing levels of As in solution. Fruit yield was decreased to 65.1, 54.1 and 37.9% in tomato plants compared to controls by the treatments 2, 5, and 10 mg As ll’, respectively. Once again, bean fruit yield was more severely influenced than tomato fruit production, with bean fruit production being reduced to 6.5 and 4.5% compared to controls at the As rates of 2 and 5 mg As 1-l) respectively. Regardless of these reductions in tomato plant growth and fruit yield, no visual symptoms of vegetative injury (chlorosis or necrosis) were observed in tomato plants throughout this experiment, and all plants were alive at the end of this study.

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Effect of arsenite concentration on dry matter production of tomato and bean plants after 36 days of treatment (plants were 64-day old after germination). Tomato and bean plants were collected at the ripening of the first fruit set As rate

Tomato Control 2mgl-’ 5mgl-’ lOmgl-’

Bean Control 2mgl-’ 5mgl-’ lOmgl-’

Dry matter production

(g plant-

Root

Stem

2.39kO.17 2.02kO.32 1.36 i 0.22 1.77 + 0.60 ***b

aa a b ab

’)

11.97+ 1.32 11.681251 6.74+ 1.56 5.OOi. 1.26 ***

Leaf

a a b b

11.53+0.97 8.88 + 1.63 4.89+ 1.04 4.15 i 0.70 ***

Fruit

a a b b

10.78 7.02 5.83 4.09 ***

+ f f +

1.06a 3.38 ab 0.62 b 1.33 b

2.82 + 0.06 a 0.60 + 0.06 b 0.58 i-O.03 b _c

5.35 f 0.08 a 1.69kO.11 b 1.77+0.15 b _

6.06 * 0.07 a 3.13 kO.07 b 1.39 f 0.07 b _

28.80* 1.54a 1.86+0.12 b 1.30+0.28 b -

***

***

***

***

aValues followed by the same letter are not significantly different ( p < 0.05). Duncan multiple range test. bNS = non significant F ratio (p < 0.05). * significant at p < 0.05, * * significant at p < 0.01, and * * * significant at p < 0.001. ‘All bean plants treated with 10 mg As(III) 1-l were dead at the end of this experiment.

Arsenic toxicity in plants was described by Machlis (1941) as consisting of root plasmolysis and leaf wilting followed by root discoloration and necrosis of leaf tips and margins. After 15 days of treatment, bean plants growing in 10 mg A&II) 1-l were wilted, stunted, with foliar chlorosis and necrosis in leaf tips and margins. Besides these visual symptoms, these bean plants took up a significant lower volume of nutrient solution than the rest of plants; nutrient solution consumption was determined by measuring the volume of solution remaining after 4 days (1 1 of solution was added to each pot every 4 days). At the end of the experiment, all bean plants growing in the nutrient solution containing 10 mg As(II1) l- ’ were dead. Wilting, stunting, chlorosis, marginal necrosis and low nutrient solution consumption seemed to imply a limitation in the water movement into the plant, though some of these symptoms can also be caused by a variety of other factors, including high concentrations of several minerals and organic compounds. Further research on transpiration, stomata1 conductance, plant water potentials and root resistance to water flux will be necessary to establish the role of the water transport in As toxicity to plants. The above described visual symptoms together with the higher plant growth and fruit yield reductions in bean plants compared to tomato plants clearly proved that arsenite was much more phytotoxic to bean than to tomato plants. 3.2. Tissue arsenic concentration Arsenic concentrations in the root of both tomato and bean plants were significantly affected by arsenite treatments (Table 2). Root As levels in both plant species increased

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Table 2 Arsenic concentration As rate

Tomato Control 2mgl-’ 5mgl-’ lOmgl-

Bean Control 2mgl-’ 5mgll’ lOmgI_

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in tomato and bean olants as affected by arsenite concentration Arsenic concentration

(mg As kg-’

Root

Stem

21.4& 1.2 aa 311.2*22.7 b 747.9 + 79.3 c 1491.2+92.5 c ***b

1.7*0.1 2.6+0.2 14.7 f 1.7 6.9 f 0.5 ***

in the nutrient solution

dry weight) Fruit

Leaf

a a b b

a a b b

0.07+0.01 0.12+0.02 0.41+ 0.06 0.24+0.06 ***

_c

2.3 +0.6 a 14.7 f 0.5 b 44.3 + 0.4 c -

5.2+0.2 a 40.6 + 0.2 b 27.2 f 0.2 c -

2.6kO.2 a 4.4kO.2 b 3.3 +0.2 b -

***

***

***

***

5.8*0.7 a 30.4+0.3 b 43.1 f 0.2 c

0X*0.1 2.7kO.2 19.9 f 4.2 18.4+ 1.4 ***

a a b b

“Values followed by the same letter are not significantly different ( p < 0.05). Duncan multiple range test. bNS = non significant F ratio (p < 0.051, * significant at p < 0.05, * * significant at p < 0.01, and * * * significant at p < 0.001. ‘All bean plants treated with 10 mg A&II) 1-l were dead at the end of this experiment.

with increasing As concentrations in the nutrient solution; this increase was proportional to the As level in solution in tomato plants (root/nutrient solution As concentration ratio was constant for all As treatments). This same ratio in bean plants decreased with increasing As levels in solution, suggesting that a restriction existed in the As uptake transport pathway. The restriction in As uptake by roots of bean plants was likely due to As damaging root cells and impairing active uptake of arsenite (Meharg and Macnair, 1991). Tomato plants accumulated As primarily in the roots. Only relatively low quantities of As were translocated to the tomato shoot (Fig. la>. On a dry weight basis, the root of tomato plants contained the highest mean As concentration with root As concentration being several orders of magnitude higher than shoot As levels. Upon As uptake by tomato plants, 83.2% (mean value of all treatments) of all the absorbed As remained in the root system, 16.8% reached the shoots and only 7.3% reached the leaves (Fig. la>. Once As has been absorbed by roots, a higher upward As translocation to the shoot was found in bean plants compared to tomato plants. Bean plants only accumulated 13.2% (mean value of all treatments) of the total plant As in the roots; 86.8% and 40.5% of the total plant As were found in shoots and leaves, respectively (Fig. lb). On a dry weight basis, roots and shoots of bean plants had similar As concentrations. The As concentrations in stems, leaves and fruit of tomato and bean plants were highly influenced by the As level in solution (Table 2). Shoot As concentration increased with increasing As levels in solution in both plant species. However, leaf and stem As concentrations of tomato plants treated with 10 mg As 1-l were lower than those of plants grown in 5 mg As 1-l; moreover, leaf As concentrations in bean plants

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treated with 5 mg As 1-l were lower than those of plants treated with 2 mg As l- ‘. This situation was likely due to the fact that the higher the As concentration in roots and stems, the more severe the toxic effects to root growth and transport functions.

100 b

&-

B

Control

m

2mgAsJL

A

5 mg As/L 0

lOmgAs/L

a -r

20

0 Root

100

m

Control

m

10mgAslL

Shdot

20

0 Robt

Shbot

Fig. 1. Total arsenic uptake (%) in roots and shoots of tomato (A) and bean (B) plants as affected by arsenite concentration. Total As uptake ( pg plant- ’ ) was calculated as the addition of the As accumulated in roots, stems, leaves, and fruit (As uptake = As concentration Xdry weight) and it was finally expressed as a percentage of the total (root + shoot) As amount accumulated by each plant.

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Therefore, As upward translocation could have been restricted by high and toxic levels of As. These results confirmed the findings of Lepp (1981) who established that extractable soil solution As levels of 5 mg As 1-l were toxic to sensitive species, such as bean. Arsenic concentrations in shoots of tomato plants have been reported to depend mainly on the root system activity (Carbonell-Barrachina et al., 1995a, Carbonell-Barrachina et al., 1995b). Upward As transport from roots to shoots is limited by its high toxicity to the radicular membranes (Wauchope, 1983). Therefore, when the As content in roots is not excessively high, there is a transport of As to shoots, and the shoot As content increased with increasing levels of As in solution. But when the As level in the root system is above a threshold level, severe damage occurs to all root cells, resulting in disruptions of root functions, restriction of As upward transport and even cellular death (Wauchope, 1983). This reduction in As translocation to shoots may result in a stabilization in the foliar As content or even in a decrease if the downward transport of As is higher than the As upward translocation (Wauchope and Street, 1987). Arsenic concentrations in tomato and bean fruit were significantly lower than the As levels in stems and leaves (Table 2). The As treatments affected fruit As concentration in a similar manner as they did As levels in leaves and stems plus branches. Berry (1986) suggested three strategies of plant tolerance to metals: avoidance (limited uptake by roots or limited transport to shoots), detoxification (subcellular compartmentalization of metal or by binding to cell walls) and biochemical tolerance (specialized metabolic pathways and enzymatic adaptations). When a toxic metal or metalloid has been absorbed by plants, the most extended mechanism involved in plant tolerance is limiting the upward transport and, resulting in accumulation primarily in the root system (Meharg and Macnair, 1991). From the data in Table 2, Fig. la, it seems that the strategy developed by tomato plants to tolerate As was avoidance; limiting As transport to shoots and increasing As accumulation in the root system. This, however, does not explain how tomato root tissue tolerates such extremely high As concentrations without exhibiting visual symptoms of toxicity. A possible explanation could be that As compartmentalization was so effective in tomato roots that As impact on growth and metabolism was minimal. Arsenic detoxification and compartmentalization in root cells are topics that will need future investigation to verify their role in plant tolerance to As toxicity. The different behavior of tomato and bean plants against arsenite pollution showed that uptake and accumulation of As in tomato and bean plants were plant species-specific. In summary, the results presented here demonstrate that the concentration of arsenite in the nutrient solution significantly affected dry matter production (phytotoxicity) and As uptake (phytoavailability) in tomato and bean plants. Arsenite was phytotoxic to both plants species; tomato plants, however, were more tolerant to As pollution than bean plants. This was likely due to the higher root-holding capacity of tomato plants to arsenic as compared to bean plants together with a very effective arsenite compartmentalization in root tissues and subcellular compartments. Bean plants exhibited symptoms of As toxicity (wilting, stunting, chlorosis, necrosis and possibly restricted water movement), and plants treated with 10 mg As 1-l were dead after 36 days of treatment. In tomato, As exposure resulted in a significant reduction in dry biomass production but

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tissue chlorosis or necrosis were not observed. Increasing levels of As in the nutrient solution resulted in higher tissue As concentrations in both plant species.

Acknowledgements We are grateful to Drs. Ronald D. DeLaune and Aroon Jugsujinda for friendly review of the manuscript. This research was supported in part by a grant from the Spanish Government (Conselleria de Educacio i Ciencia, Generalitat Valenciana).

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