Phytoextraction of Zinc by Oat (Avena sativa), Barley (Hordeum vulgare), and Indian Mustard (Brassica juncea)

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1 Environ. Sci. Technol. 1998, 32, Phytoextraction of Zinc by Oat (Avena sativa), Barley (Hordeum vulgare), and Indian Mustard (Brassica juncea) STEPHEN D. EBBS AND LEON V. KOCHIAN* U.S. Plant, Soil, and Nutrition Laboratory, USDA-ARS, Cornell University, Ithaca, New York The success of phytoremediation hinges on the selection of plant species and soil amendments that maximize contaminant removal. Indian mustard (Brassica juncea) has been shown to be effective in phytoextracting Zn, particularly after the synthetic chelate EDTA has been applied to the soil. However, the effectiveness of grass species for phytoremediation has not been addressed in great detail. A hydroponic screening of 22 grass species indicated that oat (Avena sativa) and barley (Hordeum vulgare) tolerated the high Cu, Cd, and Zn concentrations present in the solution and also accumulated elevated concentrations of these metals in the plant shoots. A hydroponic experiment comparing these two grasses to Indian mustard indicated that, although shoot Zn concentrations were greater for Indian mustard, the grasses were considerably more tolerant. A pot experiment conducted using a Zncontaminated soil showed that the addition of EDTA to the soil significantly increased Zn accumulation by B. juncea but not oat or barley. Nevertheless, barley accumulated >2 mg of Zn plant -1,2-4 times more Zn than what was observed in Indian mustard in the presence of EDTA. The results of this experiment suggest that barley has a phytoremediation potential equal to, if not greater than, that for B. juncea. Introduction The primary objective of phytoextraction is to maximize the transfer of contaminant to the plant shoots so that the greatest total mass of contaminant is removed by each cropping. Initial phytoremediation research suggested that this could be achieved with hyperaccumulator plant species such as Thlaspi caerulescens (1-4). While the results from these studies were promising, some researchers have suggested that the small size and slow growth of this species may limit its utility for phytoremediation (5, 6). Recent evidence suggests that moderate accumulating, high biomass species, such as Indian mustard (Brassica juncea), may be more effective than T. caerulescens in phytoextracting Zn (7-9). For example, when B. juncea and T. caerulescens were grown for 6 weeks in a contaminated soil [(> mg of Zn (kg of soil) -1 ], it was found that B. juncea removed 4-fold more Zn than T. caerulescens (9). This was due primarily to the fact that B. juncea produced 10 times more biomass than T. caerulescens. These results * Corresponding author phone: (607) ; fax: (607) ; lvk1@cornell.edu. suggested that a greater shoot biomass can more than compensate for a lower shoot metal concentration and that the plant species suitable for phytoremediation may not be limited to hyperaccumulators. There is evidence that grass species such as corn, barley, and ryegrass have varieties that display significant heavy metal tolerance (10, 11). Several studies (12-17) have indicated that certain varieties from the genera Agrostis, Deschampsia, Festuca, and Holcus possess a nonspecific heavy metal co-tolerance similar to that of the hyperaccumulating Thlaspi species (18, 19). That is, certain varieties display a tolerance to metals whose concentrations were not elevated in the parent soil from which the particular variety originated. Some tolerant grass species are also capable of accumulating moderate to high levels of heavy metals in the plant shoots (20-25). Because several of these grass species also produce a high biomass, the possibility exists that such species may be as effective as B. juncea in phytoextracting heavy metals. The first part of this study utilized a hydroponic screening experiment to identify grass species with the ability to tolerate and accumulate heavy metals (Cd, Cu, Zn) in plant shoots. A subsequent hydroponic experiment and a pot study using Zn-contaminated soil were then conducted to compare the phytoextraction potential of two candidate species from the screening experiment, oat (Avena sativa) and barley (Hordeum vulgare), to B. juncea. In addition, the effect of EDTA on Zn phytoextraction was examined in the pot experiment. Recent studies have shown that the removal of heavy metals can be greatly enhanced through the addition of synthetic chelates to the soil (25-27). In the study in which the effect of synthetic chelates on Zn phytoextraction was examined, however, the soil Zn concentration was achieved by spiking clean soil with zinc carbonate. Thus, there are no studies in the literature describing the effect of synthetic chelates on Zn phytoextraction from aged contaminated soil. Materials and Methods In the initial screening experiment, the grasses tested were obtained commercially: Agropyron elongatum, Agrostis tenuis, Elymus angustos, Elymus cinereus, Elymus junceus, Elymus triticoides, Eragrostis curvula, Festuca megalura, Leptochloa dubia, Pannicum virgatum, Phalaris arundinacea, Poa compressa, Poa sandbergii, Puccinellia distans (Granite Seed, Lehi, UT), Paspallum notatum, and Sorghum bicolor (Valley Seed, Fresno, CA). Two varieties each of Agrostis alba, Festuca longifolia, and Festuca rubra were also tested (Granite Seed, Lehi, UT; Big Sky Wholesale Seeds, Shelby, MT). Seed of Agrostis capillaris and a third variety of Festuca rubra were provided by Dr. David Morrey (Golder Associates, Boulder, CO). Oat (Avena sativa) and barley (Hordeum vulgare) were included for comparative purposes. Seed of Indian mustard (Brassica juncea, accessions and ), was obtained from the USDA-ARS Plant Introduction Station, Ames, IA. Grass seeds were germinated for 3-4 days on filter paper before being transferred to two 200-L polyethylene tanks containing a nutrient solution comparable to the soil solution extracted from a Zn-contaminated soil (28). A 1.25-cm-thick piece of Styrofoam, into which 100 wells (2.5 cm diameter) had been cut, was floated on the solution in each tank. Disks of polyethylene mesh (1 mm) attached to the bottom of each well supported the seeds. Within each tank, each species was replicated three times. The composition of the nutrient solution in each tank was as follows: 6.0 mm KNO 3; 4.0 mm Ca(NO 3) 2; 0.1 mm NH 4H 2PO 4; 1.0 mm MgSO 4; 25.0 µm CaCl 2; ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 6, 1998 S X(97) CCC: $ American Chemical Society Published on Web 01/27/1998

2 TABLE 1. Shoot Biomass and Removal of Heavy Metals from the Control (-) and Heavy Metal-Supplemented (+) Hypoponic Solutions during the Hydroponic Screening Experiment a biomass (g plant -1 ) Cd (µg plant -1 ) Cu (µg plant -1 ) Zn (µg plant -1 ) species Agropyron elongatum < Agrostis alba < Agrostis capillaris < < Agrostis tenuis < Avena sativa < Elymus cinereus < Elymus curvula < Elymus junceus Elymus triticoides < Festuca megalura < Festuca rubra < Hordeum vulgare Leptochloa dubia 0.05 < Phalaris arundinacea < Poa compressa 0.05 <0.01 < Puccinellia distans < a For A. alba, F. longifolia, and F. rubra, the data presented were from the best performing variety tested µmh 3BO 3; 1.0 µm MnSO 4; 1.0 µm ZnSO 4; 0.5 µm CuSO 4; 0.1 µm H 2MoO 4; and 0.1 µm NiSO 4. Chelated iron (10 µm) was provided as Fe-HEDTA, and the solution was buffered to ph 6 with 1 mm 2-(N-morhpolino)ethanesulfonic acid (MES) (Sigma, St. Louis, MO). The nutrient solution in one tank was supplemented with 100 µm ZnSO 4,5µM CuSO 4, and 1 µm CdCl 2 to simulate the metal concentrations in the soil solution from the contaminated soil. The nutrient concentrations in the tanks were monitored during the course of the experiment to ensure that the plants did not deplete the solution of essential micronutrients. Since this monitoring indicated that depletion did not occur, due to the large volume of nutrient solution in the tanks (200 L), fresh nutrient solution or metal-supplemented nutrient solution was added daily to the respective tanks to maintain the initial solution level. To avoid Fe and Mn deficiencies that may develop in plants following exposure to heavy metals (9), plants received a foliar micronutrient spray twice per week containing 1.2 mm Mn-EDTA and 7.1 mm Fe-EDDHA (as Sequestrene Fe-138, CIBA-GEIGY, Greensboro, NC). Silwet L-77 (OSI Specialties, Danbury, CT) was included as a wetting agent at a final concentration of 0.05% (v/v). After harvest, shoots were dried, digested with 1:1 (v/v) nitric and perchloric acids at 200 C, and analyzed using an inductively coupled argon plasma emission spectrometer (ICAP-ES) (Model 61E, Thermo Jarrell Ash, Franklin, MA). A hydroponic experiment was conducted to compare the phytoextraction of Zn by barley (Hordeum vulgare) and oat (Avena sativa) to that of Indian mustard (B. juncea). Seeds of each species were germinated on filter paper for 2-3 days before being transferred to 2-L pots containing the basal nutrient solution described above. B. juncea was grown at a density of two plants per pot while there were 4-6 plants per pot for oat and barley. Fe (10 µm) was provided to B. juncea as Fe-EDDHA and to the grasses as Fe-HEDTA. Plants were grown for 10 days in the nutrient solution before the Zn level in the treatment group was increased to 100 µm Zn. A subset of plants in both the control treatment groups received the foliar spray treatment described above twice per week following the appearance of toxicity or deficiency symptoms (chlorosis). Nutrient solutions were changed twice per week, and after 3 weeks, the plants were harvested and analyzed by ICAP-ES. Contaminated soil was collected from the top 12 cm of the soil profile in the vicinity of a Zn smelter in Palmerton, PA. The soil was crushed and passed through a 5-mm steel sieve. Total Zn [3100 mg (kg of dry soil) -1 ] was determined by digesting soil for 2+ hours at 180 C with a 2:1 mixture of nitric acid and hydrogen peroxide, followed by a 1:1 nitric/ perchloric acid mixture at 220 C. The ash was resuspended in 5% nitric acid and analyzed by ICAP-ES. The ph of the soil was 7.7. Seeds of oat, barley, and the two B. juncea accessions were germinated on filter paper for 2-3 days before being transferred to pots containing 2 kg of Zn-contaminated soil. Pots were watered regularly to a level close to field capacity. After emergence, plants were thinned to two per pot for all species. Beginning in the fifth week of the experiment, developing buds on B. juncea (426308) were removed to prolong the vegetative phase of development. After 8 weeks of growth, a subset of pots from each species was treated with a 25 mm solution of EDTA (tripotassium salt) to a final concentration to 2.5 g of EDTA (kg of soil) -1. The pots were placed into larger plastic pots to prevent the loss of the chelate due to leaching. Soil moisture level was maintained near field capacity for one additional week. Plants were then harvested by cutting the stems approximately 2 cm above the level of the soil. Shoots were rinsed with water to remove any soil, dried, digested, and analyzed by ICAP- ES. Three 10-cm soil cores, 5 mm in diameter, were taken from each pot at harvest. The three cores were dried, mixed to form a composite sample from each pot, and sieved through a 2-mm screen. After the ph had been measured, soil samples (5 g) were sequentially extracted with 10 ml of water to determine the effect of the EDTA on the solubility of Zn in the soil. Results After emergence, several grass species in the heavy metal treatment group became chlorotic. Most species responded favorably to the foliar spray, recovering their green color quickly. Even with foliar treatments, the impact of the heavy metals on shoot biomass production was variable (Table 1). Some species (F. longifolia, P. notatum, S. bicolor, P. sandbergii, E. argustos, P. virgatum) did not develop in the heavy metal treatment, perhaps due either to heavy metal toxicity or to slow growth in hydroponic solution. Other species grown in the presence of heavy metals produced more biomass than the control plants (E. junceus, F. megalura, E. curvula, P. distans). The increase in biomass was significant only for E. curvula (P < 0.001). In terms of heavy metal removal, oat and barley performed surprisingly well (Table 1). Barley removed more than 20 VOL. 32, NO. 6, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 803

3 FIGURE 1. Shoot Mn and Fe concentrations for oat, barley, and the two accessions of B. juncea grown hydroponically in the presence and absence of 100 µm Zn in solution. Bars denote standard error of the mean (n ) 4). times more Zn than the next best species, A. elongatum, primarily because the former produced 10 times greater biomass. The data from the heavy metal treatment for oat, however, was based upon only the single replicate that developed. In addition to A. elongatum, the species E. junceus, P. arundinacea, and E. curvula also accumulated Zn to elevated levels in the shoot. For A. elongatum and P. arundinacea, the increase in Zn was probably due to a concentration effect, as there was a 50% decrease in shoot biomass for each in the heavy metal treatment. With the possible exception of Cd accumulation by E. junceus, Cd and Cu removal by these four grass species was only marginally higher than the corresponding control plants (Table 1). In a subsequent hydroponic experiment, B. juncea plants exhibited signs of severe toxicity and nutrient deficiency when grown in the presence of elevated concentrations of Zn. Shoot Fe and Mn levels for B. juncea were in the range typically associated with deficiencies of these elements (Figure 1). B. juncea plants also showed >80% reduction in shoot biomass (Figure 2). These results are consistent with those from previous studies in our laboratory (9, 28). Oat and barley appeared to be fairly heavy metal tolerant, exhibiting little chlorosis and showing a considerably smaller reduction in shoot biomass (16 and 29%, respectively). While shoot Mn levels for oat were also in the deficiency range, shoot Fe levels for both oat and barley were similar to those in control plants (Figure 1). Of the two B. juncea accessions, only accession responded to the foliar micronutrient spray, showing a significant increase in shoot biomass. The foliar micronutrient treatment decreased the growth reduction in oat and barley to 12 and 19%, respectively (Figure 2). Shoot Zn accumulation in barley was not significantly different from that of B. juncea (Figure 3). For oat, shoot Zn accumulation was not significantly different from that of B. juncea (148290) but was significantly less than that exhibited by B. juncea (426308) (P e 0.02) in both the presence and the absence of the foliar micronutrient spray. Shoot biomass was similar for all three species in both the untreated and chelate-treated soil (Figure 4). In contrast to the hydroponic experiment, B. juncea exhibited only slight chlorosis primarily in the older leaves. Shoot Fe and Mn concentrations between the three species and between treatments were not statistically significant and were well within the adequate range (data not shown). By the fifth week of the experiment, B. juncea (426308) plants had bolted FIGURE 2. Inhibition of shoot growth due to exposure to 100 µm Zn in solution. Solid bars represent plants receiving foliar applications of chelated Fe and Mn. Control refers to the biomass produced by the respective plant species at the basal level of Zn in the nutrient solution (1 µm). FIGURE 3. Shoot Zn accumulation in control plants, plants grown on 100 µm Zn, and heavy metal-grown plants given foliar applications of chelated Fe and Mn. Bars denote the standard error of the mean (n ) 4). and flower buds had begun to develop. While pinching back these buds prolonged the vegetative phase, the plants had begun to set seed at harvest. B. juncea (148290) remained in the rosette stage until late in the experiment. While oat was free of any foliar symptoms, some barley leaves were slightly chlorotic. Oat plants in both treatment groups had set seed by the time of harvest. Barley developed seeds only in the unamended soil. The highest shoot Zn concentration and Zn removal (2 mg of Zn plant -1 ) was achieved by barley, in both the control and the EDTA treatment (Figure 5). The addition of EDTA to the soil increased Zn solubility dramatically (Figure 6). There was no effect of the chelate on the soil ph in any of the treatments. Shoot Zn concentration and accumulation increased significantly for B. juncea following the addition of EDTA (Figure 4). The grasses generally did not respond to chelate addition. Discussion The data presented here suggest that barley has a phytoremediation potential at least equal to that of B. juncea ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 6, 1998

4 FIGURE 4. Shoot biomass production for oat, barley, and the two accessions of B. juncea grown 9 weeks in Zn-contaminated soil in the presence or absence of chelate amendment. Bars denote the standard error of the mean (n ) 3). FIGURE 5. Shoot Zn concentration and total accumulation for oat, barley, and the two accessions of B. juncea grown in Zncontaminated soil for 9 weeks in the presence or absence of chelate amendment. Bars denote the standard error of the mean (n ) 3). An (*) denotes a value either significantly greater or less than the corresponding control value (P < 0.05). Throughout the study, barley consistently accumulated more Zn than B. juncea. For the hydroponic studies, differences in nutrient status between the monocots and B. juncea may have prevented the full potential of the latter species from being realized. In the pot study, however, biomass production and Fe and Mn levels for B. juncea and barley were not significantly different. Furthermore, in terms of ph and Zn solubility, the soil conditions to which each species was exposed were very similar. Under these conditions, a direct comparison between the two species can be more easily made. The response of the two species to the addition of EDTA to the soil was markedly different. The results for B. juncea are consistent with those from an earlier study (26), although the magnitude of the increase observed in the current study was somewhat smaller. This disparity may have been related to differences in Zn solubility between the soils used in the two studies. The soil used in the earlier study contained a much lower Zn concentration, 300 mg (kg of soil) -1, achieved by spiking a Sassafras AP soil with zinc carbonate (26). Although the total Zn in the Palmerton soil was considerably FIGURE 6. Soluble Zn in the amended and unamended pots of contaminated soil used for each plant species. Bars denote the standard error of the mean (n ) 3). higher (3100 mg kg -1 ), the contamination had been aged. Since there was no soil extraction data presented in this study, there is no way to determine if the level of soluble Zn differed from those observed in the current study. Despite a higher total Zn concentration, Zn solubility in the aged Palmerton soil following chelate addition may have been less than that in the spiked Sassafras AP soil. The B. juncea plants grown in the aged soil may therefore have accumulated less Zn. In contrast to the effect of the chelate on B. juncea, barley and oat did not respond to the addition of EDTA. It has been suggested that, for B. juncea, the genetic potential to accumulate metals such as Cd and Pb is not realized unless the metals are also available in the soil (26). While chelate addition overcomes this limitation for B. juncea, it does not appear to have a similar effect for the two monocots tested. Even if subsequent research shows that synthetic chelates do not induce Zn accumulation in barley, this species may still be of considerable importance for phytoremediation. As the results from the unamended soil revealed, barley accumulated Zn much more effectively than B. juncea. If this species phytoextracts Zn in the field as well as it did in this study, then this species may rival or perhaps exceed the Zn phytoextraction rate that could be achieved with field-grown B. juncea. Another approach may be to use these two species in concert, cropping the contaminated site initially or simultaneously with barley. When the point is reached where barley fails to maintain a high rate of Zn removal, chelate addition could then be used with B. juncea to solubilize residual Zn that had previously been unavailable for uptake. This approach would help to minimize the amount of chelate applied in the field, reducing the cost and alleviating the concerns that have been raised regarding the potential environmental impact of EDTA addition. In any event, the use of grass species such as barley will undoubtedly broaden the applicability of phytoremediation as well as allay the concerns of potential critics. With further research, it should be possible to maximize the efficiency of these species, making phytoremediation a more versatile tool for environmental remediation. Acknowledgments The authors would like to acknowledge the U.S. Department of Energy s Office of Science and Technology (OST) for funding this research through DOE-USDA Interagency Agreement DE-A122-95PCP5701 to L.K. The authors would also like to thank Dr. Rufus Chaney and Dr. Yin Li for their advice and assistance. VOL. 32, NO. 6, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 805

5 Literature Cited (1) Baker, A. J. M.; Reeves, R. D.; McGrath. S. P. In In situ Bioreclamation; Hinchee, R. L., Olfenbuttel, R. F., Eds.; Butterworth-Heinemann: Boston, 1991; pp (2) Baker, A. J. M.; McGrath, S. P.; Sidoli, C. M. D.; Reeves, R. D. Resour. Conserv. Recycl. 1994, 11, 41. (3) Brown, S. L.; Chaney, R. L.; Angle, J. S.; Baker, A. J. M. J. Environ. Qual. 1994, 23, (4) Brown, S. L.; Chaney, R. L.; Angle, J. S.; Baker, A. J. M. Environ. Sci. Technol. 1995, 29, (5) Black, H. Environ. Health Perspect. 1995, 103, (6) Brown, S. L.; Chaney, R. L.; Angle, J. S.; Baker, A. J. M. Soil Sci. Soc. Am. J. 1995, 59, (7) Kumar, P. B. A. N.; Dushenkov, V.; Motto, H.; Raskin, I. Environ. Sci. Technol. 1995, 29, (8) Salt, D. E.; Blaylock, M.; Kumar, P. B. A. N.; Dushenkov, V.; Ensley, B. D.; Chet, I.; Raskin. I. Biotechnology 1995, 13, 468. (9) Ebbs, S. D.; Kochian, L. V. J. Environ. Qual. 1997, 26, 776. (10) Antonovics, J.; Bradshaw, A. D.; Turner, R. G. Adv. Ecol. Res. 1971, 7, 1. (11) Baker, A. J. M. New Phytol. 1987, 106 (Suppl.), 93. (12) Symeonidis, L.; McNeilly, T.; Bradshaw, A. D. New Phytol. 1985, 101, 309. (13) Herstein, U.; Jäger, H.-J. Environ. Exp. Bot. 1985, 26, 309. (14) Coughtrey, P. J.; Martin, M. H. New Phytol. 1978, 81, 147. (15) von Frenckell-Insbam, B. A. K.; Hutchinson, T. C. New Phytol. 1993, 125, 547. (16) von Frenckell-Insbam, B. A. K.; Hutchinson,T. C. New Phytol. 1993, 125, 555. (17) Patra, J.; Lenka, M.; Panda, B. B. New Phytol. 1994, 128, 165. (18) Reeves, R. D.; Baker, A. J. M. New Phytol. 1984, 98, 191. (19) Baker, A. J. M.; Reeves, R. D.; Hajar, A. S. M. New Phytol. 1994, 127, 61. (20) Reilly, A.; Reilly, C. New Phytol. 1973, 72, (21) Brooks, R. R.; Reeves, R. D.; Baker, A. J. M.; Rizzo, J. A.; Ferreira. H. D. Nat. Geogr. Res. 1989, 6, 205. (22) Frossard, R.; Stadelmann, F. X.; Niederhauser, J. J. Plant Physiol. 1989, 134, 180. (23) Gerzabek, M. H.; Schaffer, K. Bodenkultur 1989, 40, 195. (24) Brown, G.; Brinkmann, K. Plant Soil 1992, 143, 239. (25) Huang, J. W.; Cunningham, S. D. New Phytol. 1996, 134, 75. (26) Blaylock, M. J.; Salt, D. E.; Dushenkov, S.; Sakharova, O.; Gussman, C.; Kapulnik, Y.; Ensley, B. D.; Raskin, I. Environ. Sci. Technol. 1997, 31, 860. (27) Huang, J. W.; Chen, J.; Berti, J. R.; Cunningham, S. D. Environ. Sci. Technol. 1997, 31, 800. (28) Ebbs, S. D.; Lasat, M. M.; Brady, D. J.; Cornish, J.; Gordon, R.; Kochian, L. V. J. Environ. Qual. 1997, 26, Received for review August 6, Revised manuscript received November 19, Accepted December 5, ES970698P ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 6, 1998