Comparative Lead Uptake and Responses of Some Plants

Grown on Lead Contaminated Soils

Gregorio B. Begonia

Department of Biology, Jackson State University, Jackson, MS 39217

The feasibility of using vegetation as a viable, cost-effective alternative to clean up metal-contaminated soils depends largely on the identification and selection of plant species that possess the ability to accumulate metals, while producing high biomass using current crop production and management practices. Therefore, this experiment was conducted to compare five plant species in terms of their ability to tolerate toxic levels of lead (Pb) and accumulate lead in shoots and roots. Plants were grown outdoors under natural daylight in artificial soil media (perlite:vermiculite) containing 0 or 500 µg/ml lead. Results indicated that the five plant species tested exhibited differential sensitivity to toxic levels of lead. Based from the magnitudes of reduction in leaf area and dry tissue biomass, radish was the most sensitive while black mustard was the most tolerant to lead. Among the five species evaluated, sunflower had the greatest ability to accumulate lead in the roots, but it translocated the least amount to its shoots. On the other hand, morningglory absorbed the smallest amount of lead in its roots, but it was the most efficient translocator of lead to its shoots. Sunflower is the most suitable species for phytoextraction of soil lead if the whole plant biomass is harvested. However, morningglory is the preferred species if only the above-ground biomass is harvested. The high lead accumulation by both sunflower and morningglory suggests that these plants should be evaluated further for their potential use in cleaning up toxic metals from contaminated soils.

Plants serve as primary source of carbon for the remainder of life forms on earth and, as such, can act as vectors for contaminant introduction into the food chain. Considering their relative position in many natural food chains, metal accumulating plants are directly or indirectly responsible for a large proportion of the dietary uptake of toxic heavy metals by humans and animals. Although some heavy metals are required for life's physiological processes (e.g., components of metalloenzymes), their excessive accumulation in living organisms is always detrimental. Generally, toxic metals cause enzyme inactivation, damage cells by acting as antimetabolites or form precipitates or chelates with essential metabolites. The impact of some toxic metals on human health has been reported by Forstner (1995). For example, a continuous oral intake of 200 µg cadmium/day could cause an increased prevalence of kidney damage in persons over 50 years of age. Consumption of selenium-laden plants was reported to be the cause of "alkali disease and blind staggers" in livestock (Banuelos and Meek, 1990; Davis, 1986). The threat that heavy metals pose to human and animal health is aggravated by their long-term persistence in the environment. For instance, lead (Pb), one of the more persistent metals, was estimated to have a soil retention time of 150 to 5000 years (Shaw, 1990). Also, the average biological half-life of cadmium, another "accumulation poison" similar to lead, has been estimated to be about 18 years (Forstner, 1995).

Heavy metals are present in soils as a consequence of human activity. Metal-rich mine tailings, metal smelting, electroplating, battery recycling, wood treating, fuel burning and fuel production, downwash from power lines, intensive agriculture and sludge dumping are the most important human activities that contaminate soils with large quantities of metals (Moffat, 1995; Forstner, 1995). In 1986 there were approximately 1,000 national priority or superfund sites in the United States that pose significant environmental or health risks. About 40% of these sites reported metal problems. The metals often cited as a problem are lead, cadmium, chromium and arsenic (Forstner, 1995). In spite of the ever-growing number of toxic metal contaminated sites, the most commonly used methods dealing with heavy metal pollution are either the extremely costly process of excavation and burial or simply isolation of the contaminated sites. Such cleanup is practical only for small areas, often a half hectare or less, and cleaning one hectare to a depth of one meter costs between 600 thousand and 3 million dollars depending on the type and intensity of pollution (Moffat, 1995).

In recent years, however, the value of metal-accumulating plants for environmental cleanup has been vigorously pursued (Taylor et al., 1992; Brown et al., 1995), giving birth to a specific area of phytoremediation termed phytoextraction (Kumar et al., 1995). The process of phytoextraction generally requires the translocation of heavy metals to easily harvestable shoots. In some cases, roots and other subterranean organs can be harvested as well. In the phytoextraction process, several hyperaccumulating plants may be used in a cropping scheme to reduce soil concentrations of heavy metals to environmentally acceptable levels.

There are many advantages of using metal-scavenging plants for the removal of metals from contaminated soils. Depending on the species, such metal-accumulating plants can be grown in multiple succession over several areas in a given year, and they are usually inexpensive to produce. For instance, Banuelos and Meek (1990) showed under field conditions that when brown mustard (Brassica juncea L.) was planted over several years and managed with minimal irrigation, selenium levels were reduced up to 50% within a soil depth of one meter. An added benefit may come from the fact that the harvested plants can be burned and the metal, such as nickel, can be recovered. Aside from lower costs and generation of recyclable metal-rich plant residues, the other benefits of using plants to clean up metal-laced soils include wide applicability to a range of toxic metals and radionuclides, minimal environmental disturbance, elimination of secondary air- or water-borne wastes, and public acceptance.

This project's objective, to develop better phytoremediation techniques, initially focused on the identification of suitable crop and plant species that showed the ability to accumulate heavy metals while producing large amounts of biomass using established agricultural practices. Particular emphasis was placed on some edible members of the Brassicaceae family, as well as on other crop species related to many wild species known to be metal accumulators. Lead was used in the study because of its importance and persistence as an environmental pollutant. Specifically, the objectives of the project were to: (a) identify through pot studies those outdoor-grown plants that demonstrate vigorous growth in heavy metal-contaminated soils, and (b) select those plants which take up high levels of heavy metals, preferably in either modified organs, if any, or which translocate heavy metals to above-ground portions of the plant.

MATERIALS AND METHODS

Plant Materials--Seeds of Indian mustard [Brassica juncea (L.) Czern.], black mustard [Brassica nigra (L.) Koch cv. Giant Southern Curled], radish (Raphanus sativus L. cv. Sparkler), sunflower (Helianthus annuus L . cv. Mammoth) and morningglory (Ipomea triloba L.) were obtained from local seed markets.

Plant Culture and Experimental Design--Plants were grown outdoors under natural daylight. Unless otherwise specified, seeds were sown in 1 liter plastic pots containing equal volumes of horticultural grade, coarse perlite and vermiculite. Based from a preplanting germination test for each species, a predetermined number of seeds were planted per pot. Emerged seedlings were thinned out to a desired population density at ten days after planting. Two concentrations (0 and 500 µg/ml) of lead (supplied as aqueous solutions of lead nitrate) were applied to the surface of the growth medium beginning at two weeks after planting and once a week thereafter until harvest. Plants were watered every other day with full strength, modified nutrient solution (Triplett, Blevins and Randall, 1980). Lead nitrate and nutrient solutions were applied on different days during a given week to prevent possible precipitation of lead by the sulfate and phosphate components of the nutrient solution. For each plant species, treatments were arranged in a Completely Randomized Design (CRD) with 4 replications. Ten pots of plants constituted a replicate of a treatment. Excess soil moisture draining from perforations at the bottom of each pot was trapped in a 10-cm plastic saucer placed below each pot to prevent leaching into the soil and cross contamination among pots. Whenever rain was imminent, plants were covered with a clear polyethylene film that was secured on a chicken wire and placed 75 cm above the plants.

Any metal toxicity symptoms (e.g., yellowing, stunting) exhibited by plants were visually noted during the experimental period. Just before harvest, total leaf area for each plant was measured. Subsamples of twenty 0.38 cm² leaf discs were randomly obtained from various leaves of each plant using a 0.7 cm diameter cork borer. The specific leaf area (leaf disc area/leaf dry weight ratio, cm²/g) of the leaf discs were used to convert total leaf weight into total leaf area. Three weeks after the initial lead treatment, all plants were harvested. Shoots and roots were separated, washed with tap water to remove any adhering debris, then oven-dried at 70C for at least 48 hours. Dried samples were weighed and ground in a Wiley mill equipped with a 425-µm (40 mesh) screen.

Heavy Metal Analyses--Lead contents of each tissue were extracted using previously described nitric acid-hydrogen peroxide procedures (USEPA, 1990) with slight modifications. Briefly, 40 ml of 50% aqueous nitric acid were added to a representative 1- to 2-gram sample of ground plant tissue. The acidified sample was heated to 95C, refluxed for 15 minutes without boiling and then allowed to cool. Another 10 ml of 50% aqueous nitric acid were added and the sample was again heated and refluxed for 30 minutes. The heated sample was allowed to cool, then completely oxidized in 5 ml concentrated nitric acid. The oxidized solution was allowed to evaporate to approximately 5 ml without boiling. To initiate the peroxide reaction, 2 ml of deionized, distilled water and 3 ml of 30% hydrogen peroxide were added to the concentrated digestate and then was heated until effervescence subsided. Another 7 ml of 30% hydrogen peroxide were added continuously in 1 ml aliquots as the digestate was again heated. The digestate was heated until effervescence was minimal and its volume reduced to approximately 5 ml. After cooling, the final digestate was diluted to about 100 ml with deionized, distilled water. The digestate was filtered through a filter paper (Whatman No. 1) and the final volume was adjusted to 100 ml with deionized, distilled water.

Lead contents of each sample were quantified using inductively coupled argon plasma spectroscopy (Perkin Elmer Optima 6000) and expressed as µg lead/gram dry weight of plant tissue.

RESULTS

Generally, all lead-treated plants showed reduced leaf area expansion

Shoot growth in radish and Indian mustard was inhibited by 52% and 35%, respectively, in plants with lead added to soil. Sunflower was least affected by lead, with a shoot biomass reduction of 6% (Fig. 2).

Roots of all lead-treated plants were purplish, in contrast to the dirty white roots of untreated plants (data not shown). Aside from the visual differences in color, root growth of all lead-treated plants was retarded, compared to the controls (Fig. 3). Radish showed the greatest sensitivity to lead, showing a 45% reduction in root biomass. Black mustard, on the other hand, exhibited the least sensitivity to lead.

All tested species concentrated lead in their shoots (Fig. 4A). The amount of lead translocated to the shoots (µg Pb/g dry tissue) of each species was: sunflower (232), Indian mustard (407), black mustard (419), radish (625), morningglory (686). The concentrations of lead accumulated in the roots (Fig. 4B) by species were: morningglory (1020), Indian mustard (2542), radish (3117), black mustard (3794), and sunflower (4391). Among the five species tested, sunflower accumulated the greatest amount of lead in its roots, but it translocated the least amount to its shoots. On the other hand, morningglory absorbed the smallest amount of lead in its roots, but it was the most efficient translocator of lead to its shoots. In all five species, the concentration of lead was higher in the roots than in the corresponding shoots. On average, the amount of lead accumulated in the roots was eight times greater than in the shoots.

DISCUSSION

The success of phytoextraction in an environmental cleanup effort depends to a large degree on the identification of suitable plants that not only concentrate metals to levels that would inhibit growth of most species, but demonstrate prolific growth in response to an established agronomic or horticultural practice. Such prolific growth produces the necessary biomass to extract large amounts of metals per hectare that are commonly encountered in most contaminated sites.

Stunting is a commonly observed growth response in a wide range of plants grown in metal-laden soils (Foy, Chaney and White, 1978). The stunting or reduced shoot biomass (Fig. 2) and decreased root biomass of lead-treated plants can be due to a specific toxicity of the metal to the plant, antagonism with other nutrients in the plant, or inhibition of root penetration in the soil. In this study, root penetration was not hindered since plants were grown in relatively small volumes of porous, artificial soil media. Although the nutrient solution and aqueous lead nitrate were applied separately, it is possible that the stunting and anthocyanin pigmentation in leaves of lead-treated plants can be ascribed to a deficiency of an element like phosphorous. Lead had been shown to form insoluble complexes with phosphorous (Johnson and Proctor, 1977; Johnson, McNeilly and Putwain, 1977). Similar anthocyanin pigmentation and inhibited growth have been recently noted in a corollary greenhouse study involving Indian mustard treated with 500 µg/ml lead (Daniels-Davis, 1996). In that study, shoot and root biomass of lead-treated plants were reduced 6% and 44%, respectively compared to the untreated controls. The differential growth responses of the various species to lead suggest that phytotoxic mechanism of lead involve different biochemical pathways in different plant species. The exact nature of these mechanisms was not investigated in this study, but is currently being pursued in our laboratory.

Results from this study demonstrated that although all five species accumulated lead, they exhibited differential ability to take up lead from solid media (e.g., perlite/vermiculite) and to transport and concentrate lead in their shoots (Fig. 4). Although sunflower accumulated the greatest amount of lead in its roots, it was least effective in translocating lead to its shoots. The metal-scavenging ability of the three Brassica species is not surprising since some wild species in the Brassicaceae family have been found to grow on metalliferous soils and accumulate large amounts of heavy metals in their roots and shoots (Baker, 1981; Baker and Brooks, 1989). It must be noted however, that although radish showed the ability to accumulate lead, its sensitivity to high levels (500 µg/ml) of lead as evidenced by its reduced biomass, may limit its potential in any phytoextraction effort.

Tight binding of lead to soils and plant material at least partially explains the relatively low mobility of this metal in soils and plants. Lead binding to clay and organic matter and its inclusion in insoluble precipitates make a significant fraction of lead unavailable for root uptake by field-grown plants. While plants are known to concentrate lead in the roots, lead translocation to the shoots is normally very low (Jones, Clement and Hopper, 1973; Jones, Jarvis and Cowling, 1973; Malone, Koeppe and Miller, 1974). Actively growing roots provide a barrier which restricts the movement of lead to the above-ground parts of plants (Jones, Clement and Hopper, 1973; Jones, Jarvis and Cowling, 1973). This restricted movement of lead may explain why lead concentrations in shoots were relatively less than in the roots. This point of view is further substantiated by a recent finding which showed that significant lead translocation to the shoots of Indian mustard was observed only at relatively high concentrations of lead in the hydroponic solution and after the lead-binding capacity of roots was partially saturated (Kumar et al., 1995).

IMPLICATIONS AND FUTURE RESEARCH

(1) Among the five species evaluated, sunflower is the most suitable species for phytoextraction of lead from soil if the whole plant biomass is harvested. However, morningglory is the preferred species if only the above-ground biomass is harvested.

(2) This study was done using artificial soil media (perlite and vermiculite), where the actual amount of the metal available to the plant could be different from that of a soil existing in a metal-contaminated site. Therefore, caution must be exercised in extrapolating such results to field conditions. Also, the results may not reflect field fluctuations in soil moisture and nutrient levels, as well as competition from other species growing in the metal-contaminated site.

(3) This preliminary study must be extended to evaluate a greater number of plant species and eventually to select those species that possess the ability to tolerate and accumulate toxic metals. Preferably, vegetative planting materials or seeds should be collected from plants growing in known metal-contaminated sites. (Permission to enter such metal-contaminated sites could be a problem).

(4) Hyperaccumulator plant species identified from preliminary studies will be further evaluated for their growth tolerance and metal uptake attributes using actual soils (if possible) from metal-contaminated sites. By quantifying metal levels of soils and plant metal uptake before and after harvest, the actual effectiveness of a plant for cleaning up metal-contaminated soils can be determined.

ACKNOWLEDGMENTS

This research was supported through a Faculty Summer Research Award from the Mississippi Urban Research Center, Jackson State University, Jackson, MS. The author would like to thank Dr. John D. Hesketh, Plant Physiologist, Photosynthesis Research Unit, USDA-ARS, University of Illinois, Urbana, IL 61801 for his critical review of the manuscript.

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