Environ. Sci. Technol. 2001, 35, 3237-3241
Hyperaccumulation of Zn by Thlaspi caerulescens Can Ameliorate Zn Toxicity in the Rhizosphere of Cocropped Thlaspi arvense S T E V E N N . W H I T I N G , †,§ JONATHAN R. LEAKE,† STEVE P. MCGRATH,‡ AND A L A N J . M . B A K E R * ,†,§ Department of Animal and Plant Sciences, The University of Sheffield, Sheffield, S10 2TN, U.K., and Agriculture and Environment Division, IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, U.K.
The metal hyperaccumulating plant Thlaspi caerulescens is effective in depleting plant-available metals from the soil. We hypothesized that this reduction of toxic metals in the rhizosphere of T. caerulescens would increase the growth of less metal-tolerant plants with their roots permitted to intermingle and develop coincident rhizospheres. The extent of rhizosphere interaction between T. caerulescens and a coplanted nonaccumulator species, Thlaspi arvense, was controlled using barriers. Two media with elevated concentrations of water-extractable Zn were prepared by enriching one soil with zinc oxide (ZnO) or zinc sulfide (ZnS). The shoot mass of T. arvense was increased by 30% when its roots were permitted to intermingle with those of T. caerulescens in the ZnO treatment. The concomitant 2-3fold reduction in shoot Zn concentration in T. arvense confirmed that its improved growth was associated with reduced uptake and phytotoxicity of Zn. Thlaspi arvense also showed increased growth and reduced metal uptake when cocropped with T. caerulescens in the ZnS treatment. We conclude that the strong Zn accumulation by T. caerulescens might enhance the establishment and development of surrounding less-tolerant species on soils that are naturally- or anthropogenically-enriched with metals.
Introduction Metal hyperaccumulating plants show an unusually high uptake of specific metals from the soil (1). For example, the crucifer Thlaspi caerulescens J. & C. Presl hyperaccumulates zinc (Zn) and cadmium (Cd) to >10 000 mg kg-1 and >100 mg kg-1 shoot dry weight, respectively, when growing on soils that have elevated concentrations of these metals (2, 3). Thlaspi caerulescens has been shown to possess a number of adaptive traits that enhance the rate (4-7) and efficiency (8, 9) with which it is able to acquire metals from soil. This scavenging of metals by roots of T. caerulescens rapidly * Corresponding author phone: +61 3 8344 5055; fax: +61 3 9347 5460; e-mail:
[email protected]. † The University of Sheffield ‡ Agriculture and Environment Division, IACR-Rothamsted. § Current address: School of Botany, The University of Melbourne, Parkville, VIC 3010, Australia. 10.1021/es010644m CCC: $20.00 Published on Web 06/28/2001
2001 American Chemical Society
depletes the concentrations of plant-available metals in soil (8, 10, 11). Although vegetation on metal contaminated soils can be sparse, hyperaccumulator plants are rarely present in monoculture and, indeed, often grow alongside with nonaccumulator species (3). The effects of high metal uptake by T. caerulescens on surrounding or cocropped nonaccumulator plants have never been tested. We hypothesized that the efficient removal of bioavailable and hence phytotoxic metals from soil solution by T. caerulescens might enhance the vigor of coplanted lesstolerant species. There has been considerable interest in the use of metal-accumulating plants for the phytoextraction of metals from contaminated land (1, 12), where the hyperaccumulator plants can then be harvested to recover the metals. Here, we are more interested in the immediate effects of metal uptake by hyperaccumulator plants on the toxicity of the soil to cocropped species. In other words, the metal removal by the hyperaccumulating plants might aid the establishment of other species that would otherwise find the metals in the soil to be toxic and inhibiting to growth. If there is a positive effect on the growth of the nonaccumulator species, cocropping with hyperaccumulators might be applied to enhancing the rate and efficiency of the revegetation of contaminated soils with less tolerant species (“phytoprotection”). The hypothesis was tested by controlling the extent of root interaction between the hyperaccumulator and nonaccumulator plants using vertical barriers: (i) no barrier, permitting complete root intermingling; (ii) roots separated, but soil solution exchange permitted via a mesh barrier that divided the pot vertically in to two halves; (iii) complete separation of the pot by a solid, impermeable barrier to prevent root and soil interactions between the two compartments. The nonaccumulator Thlaspi arvense L. has a much lower threshold of tolerance to Zn than T. caerulescens and was used as the companion species. Thlapsi arvense does not naturally co-occur with T. caerulescens.
Materials and Methods Construction of the Pots. The pots were constructed from 5 cm lengths of 4 cm diameter poly(vinyl chloride) (PVC) pipe, each with a nylon mesh base (35 µm pore size). Three pot types were prepared: One was undivided (no central barrier). The control pot was compartmentalized into two halves by a vertical barrier of PVC. The third pot was intermediate, with a vertical nylon mesh barrier (35 µm pore size). The central barriers and mesh base were glued in place using Polypipe Cement (Polypipe Plc, U.K.), and the pots were “cured” in a ventilated fan oven at 45 °C for 4 weeks to eliminate the highly phytotoxic solvent in the cement. Zn Enrichment of the Soil. The sandy-loam soil was collected from a well-tilled agricultural site at Woburn, U.K. This was subsequently enriched to nominal concentrations of 1000 mg Zn kg-1 with either ZnO or ZnS (9). Zinc oxide was selected as one source of Zn because it has limited solubility in water (1.6 mg L-1 in cold water (13)); this has the advantage of avoiding excessively high water-extractable Zn concentrations that would otherwise kill the T. arvense plants, while enabling the supply of a large amount of Zn to replenish that depleted from the soil solution via uptake by T. caerulescens. ZnS was used as the second source of Zn because it was found to provide a lower concentration of water-extractable-Zn than ZnO when added to Woburn soil (Table 1). The ZnS treatment was therefore less toxic to the T. arvense plants. The concentrations of total Zn and waterextractable Zn in soils from both soil treatments (Table 1) VOL. 35, NO. 15, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Experimental design for the nine treatments of coplanted Thlaspi caerulescens and T. arvense. H ) Zn-hyperaccumulating T. caerulescens; N ) nonaccumulating T. arvense.
TABLE 1. Concentrations of Total-Zinc, Total-Cadmium, and Water-Extractable-Zn (mg kg-1) and the pHs of the Soils after Equilibration at 60% WHCa soil treatment
total Zn
WE-Zn
total Cd
pH (H2O)
ZnO ZnS tailings
1002 ( 117 1073 ( 58† 1676 ( 102‡
4.28 ( 4.39 0.61 ( 0.10 NA
NA NA 39.5 ( 3.2‡
7.30 6.60 6.38
a Data are means ( standard errors (n ) 5, except † where n ) 4, and ‡ where n ) 6). Analyses were performed using methods given in ref 9. WE-Zn - water extractable Zn; NA - not assayed.
were measured after equilibration for 14 days in a controlled environment room (16-h, 20 °C, 183 µmol m-2 s-1 photon flux density day; 8-h, 14 °C night) at 60% water holding capacity (WHC). Extraction with water was used as an estimate of the concentration of soluble-Zn in the soils. The pH of moist soil samples (10 g) was determined in slurry of 1:2.5 soil:water (Table 1). Cocropping in ZnO Treated Soil. Nine treatments were prepared to investigate the effects of three levels of root interaction on the shoot mass and Zn accumulation by T. caerulescens and T. arvense planted in mono- or mixedcultures (Figure 1). Seventy grams of the ZnO-treated soil was added to each of 45 pots, wetted to 60% WHC, and allowed to equilibrate in the controlled environment room for 14 days. Four seedlings of T. caerulescens were planted into each half of the pots as indicated by “H” in Figure 1. Seven days later, two seedlings of the nonaccumulator T. arvense were transplanted to each side of the pots as indicated by “N” in Figure 1. This gave either a monoculture of T. caerulescens, a monoculture of T. arvense, or a mixed culture of T. caerulescens on one side of the pot and T. arvense on the other. Each pot/species combination was replicated five times. The seeds of T. caerulescens were from the Prayon population, Belgium, and the seeds of T. arvense were from the Crucifer Genetics Cooperative, WI. The pots were randomized in the controlled environment room, and water was added to return the soils to 60% WHC as required. Plants were harvested 42 days after the T. caerulescens were transplanted into the pots. Plants on each side of the pots were harvested, dried at 80 °C, and analyzed separately: The dry shoots were weighed and digested by wet-ashing with H2SO4/H2O2 at 380 °C. The Zn concentrations in the digest solutions were determined by flame Atomic Absorption Spectroscopy (AAS). Cocropping in ZnS Treated Soil. Seventy grams of the ZnS-treated soil was placed in each pot instead of ZnO-treated soil. The ZnS treatment was prepared in exactly the same way as the ZnO treatment: The 45 pots were planted with 3238
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FIGURE 2. Thlaspi arvense in ZnO-treated soil: shoot mass, concentration of Zn, and total Zn accumulated per shoot in T. arvense from pots containing a mixed-culture of coplanted T. caerulescens and T. arvense (hatched bars) and from one side of pots containing a monoculture of T. arvense (gray bars). The three levels of root interaction (no barrier, mesh barrier, and full barrier) are indicated using the symbols shown in Figure 1. Data are presented as means + standard errors, n ) 5. Significant ANOVA are shown by letter codes; values showing the same letter are not significantly different (Tukey multiple comparisons test, P < 0.05). mixtures- or monocultures of T. caerulescens and T. arvense (Figure 1), grown, harvested, and analyzed as described above. Statistical Analyses. ANOVA were used to determine differences between the treatments (i.e., mixed- or monoculture, and the extent root intermingling permitted) on the shoot mass, Zn concentration, and mass of Zn accumulated in each species. Inspection of the data sets indicated that a few failed the requirements of ANOVA (14); transformations were performed where necessary.
Results and Discussion Cocropping caused increases in the growth of both species where unrestricted root intermingling was permitted between T. arvense and T. caerulescens. The ZnO-treated soil contained a high concentration of water-extractable Zn (Table 1; 4.28 mg kg-1), and the leaves of T. arvense showed chlorosissa symptom of Zn toxicity. Cocropping with T. caerulescens, however, ameliorated the toxic effects of Zn on T. arvense. In monocultures, the shoots of T. arvense attained the same biomass irrespective of the extent of root intermingling permitted within the pots (Figure 2a, gray bars). Similarly, the biomass of shoots of T. arvense remained unaffected by T. caerulescens where the intermingling of their roots was prevented by solid or mesh barriers. In contrast, the shoot biomass of T. arvense was increased by 30% where root intermingling with T. caerulescens was permitted (Figure 2a; P < 0.05). This growth enhancement of T. arvense appeared to be the result of reduced concentrations of Zn in the shoots when its roots intermingled with those of T. caerulescens. When grown in monoculture, the concentration of Zn in the shoots of T. arvense was constant, irrespective of the extent of root intermingling permitted (Figure 2b, gray bars). These concentrations (∼1500 mg kg-1) are almost 5 times the concentration of Zn considered as a threshold of toxicity for many dicotyledonous species (>300 mg kg-1 (15)) and hence retarded the growth of T. arvense. In mixed-culture with T. caerulescens in the pots without barriers however, the concentration of Zn in the shoots of T. arvense (500 mg kg-1)
FIGURE 3. Thlaspi arvense in ZnS-treated soil: shoot mass, concentration of Zn, and total Zn accumulated per shoot of the nonaccumulator T. arvense from pots containing a mixed-culture of coplanted T. caerulescens and T. arvense (hatched bars) and from one side of pots containing a monoculture of T. arvense (gray bars) growing in ZnS-treated soil. The three levels of root interaction (no barrier, mesh barrier, and full barrier) are indicated using the symbols shown in Figure 1. Data are presented as means + standard errors, n ) 5. Significant ANOVA are shown by letter codes; values showing the same letter are not significantly different (Tukey multiple comparisons test, P < 0.05). was only a third of that seen in plants grown in pots with barriers, a difference which was significant (Figure 2b, hatched bars; P < 0.05). Furthermore, the total mass of Zn accumulated by T. arvense was lower in the treatment where full root interaction with the hyperaccumulator was permitted than in any of the other treatments (Figure 2c). Thlaspi arvense showed the same growth responses when cocropped with T. caerulescens in the ZnS-treatment. The total concentration of Zn in the soil in the ZnS-treated soil was the same as in the ZnO treatment, but the concentration of extractable-Zn was much lower (Table 1). Here the shoots of T. arvense did not show any chlorosis, which indicates that the ZnS-treatment was not chronically toxic to T. arvense. The shoots of T. arvense in this treatment still showed improved growth when cocropped with T. caerulescens. The shoot mass of T. arvense was greatest where root intermingling was permitted with T. caerulescens (Figure 3a), but this was not significant (P > 0.05). The accumulation of Zn in the shoots of T. arvense when sharing a complete rhizosphere with T. caerulescens was also less than in the other treatments (Figure 3b,c), as seen in the ZnO treatment. The hyperaccumulator plant T. caerulescens showed improved growth in the pots where intermingling with the nonaccumulator species was permitted. In the ZnO treatment, the shoot mass of T. caerulescens was constant when grown in monoculture, irrespective of the extent of root intermingling permitted (Figure 4a, black bars). When grown in mixed culture with T. arvense (hatched bars), the shoot mass of T. caerulescens was increased by approximately 15% where limited root interaction was permitted between the two species by a mesh barrier (not significant, P > 0.05) and by 39% in the pots without barriers, which was significant (P < 0.05). The same growth responses were seen for T. caerulescens in the ZnS treatment (Figure 5a). These positive effects on the shoot mass of T. caerulescens when sharing its rhizosphere with T. arvense suggest that T. caerulescens showed greater intensity of competition in monoculture than when grown at the same density in mixed culture. When
FIGURE 4. Thlaspi caerulescens in ZnO-treated soil: shoot mass, concentration of Zn, and total Zn accumulated per shoot in T. caerulescens from pots containing a mixed-culture of co-planted T. caerulescens and T. arvense (hatched bars) and from one side of pots containing a monoculture of T. caerulescens (black bars) growing in ZnO-treated soil. The three levels of root interaction permitted (no barrier, mesh barrier, and full barrier) are indicated using the symbols shown in Figure 1. Data are presented as means + standard errors, n ) 5. Significant ANOVA are shown by letter codes; values showing the same letter are not significantly different (Tukey multiple comparisons test, P < 0.05).
FIGURE 5. Thlaspi caerulescens in ZnS-treated soil: shoot mass, concentration of Zn, and total Zn accumulated per shoot in T. caerulescens from pots containing a mixed-culture of coplanted T. caerulescens and T. arvense (hatched bars) and from one side of pots containing a monoculture of T. caerulescens (black bars) growing in ZnS-treated soil. The three levels of root interaction permitted (no barrier, mesh barrier, and full barrier) are indicated using the symbols shown in Figure 1. Data are presented as means + standard errors, n ) 5. Significant ANOVA are shown by letter codes; values showing the same letter are not significantly different (Tukey multiple comparisons test, P < 0.05). grown in mixed culture, T. caerulescens was benefiting from reduced competition for resources in the pot. Indeed, T. caerulescens showed increased Zn accumulation in this treatment (Figures 4c and 5c). One explanation for the increased growth of the nonaccumulator, T. arvense, when sharing a rhizosphere with T. VOL. 35, NO. 15, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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caerulescens is that it too may show greater intensity of competition in monoculture than when grown at the same density in mixed culture. However, we believe this effect is unlikely in these soils. In a separate study using a low-Zn soil (21 mg Zn kg-1), the two species of Thlaspi were cocropped under exactly the same conditions as in the experiments presented here (16). In that low-Zn soil, the plants of T. arvense attained a biomass of ∼40 mg, over two times the biomass attained by T. arvense in the present study. The smaller biomass of T. arvense in the present study was a result of the toxicity of the high concentration of Zn in the soil. It is unlikely that T. arvense would show highly competitive abilities when suffering such Zn toxicity. The results are consistent with the hypothesis that T. caerulescens diminished the pool of water-soluble Zn in the soil that was otherwise phytotoxic to T. arvense. In the shared rhizosphere treatment T. arvense was better able to tolerate the reduced concentration of soluble-Zn, and consequently, its shoot growth was enhanced. There are two possible mechanisms leading to the reduction in the concentration of water-soluble Zn in the rhizosphere of T. caerulescens: (i) the fast rate of Zn uptake and translocation by this species (4-7) rapidly depleted the concentration of soluble-Zn in the soil or, alternatively, (ii) T. caerulescens reduced the concentration of soluble-Zn in the soil by immobilizing it in the rhizosphere, e.g., by precipitation/chelation with compounds exuded from the roots (15). The latter of these two mechanisms is improbable since the extremely high uptake of Zn by T. caerulescens means that it is more likely to mobilize Zn in the rhizosphere than to immobilize it (8, 11). The data from our experiments support the first mechanism, the removal of soluble-Zn from the soil by accumulation in T. caerulescens. The first line of evidence comes from the total mass of Zn accumulated in the shoots of T. caerulescens. In both the ZnO and ZnS treatments, T. caerulescens accumulated more Zn when root intermingling with T. arvense was unrestricted in comparison with the other five pot/species treatments (Figures 4c and 5c). These increases indicate that the hyperaccumulator was depleting soluble-Zn from the soil around T. arvense. The observed increases in Zn accumulation by T. caerulescens in pots where mesh barriers separated the two species of Thlaspi also supports this conclusion; T. caerulescens removed solubleZn from its compartment, plus some from the T. arvense side of the pot by transpiration-driven mass flow through the mesh. This is consistent with the findings of McGrath et al. (8) who showed that T. caerulescens decreased the concentration of NH4NO3-extractable Zn in soil on both sides of a mesh barrier. The second line of evidence to support our conclusion that the strong accumulation of Zn by T. caerulescens was depleting the phytotoxic-Zn comes from estimates of the “size” of the pools of soluble-Zn in the pots. From the concentration of water-extractable Zn in the ZnO-treated soil (Table 1), we can estimate that the 70 g of soil in each pot contained 300 µg of soluble-Zn. This is almost exactly the mass of Zn accumulated by the T. caerulescens (Figure 4c, 280 µg) plus T. arvense (Figure 2c, 7 µg) plants in mixedculture with roots intermingling. Moreover it is close to the mass of Zn accumulated by T. caerulescens grown in monoculture in this treatment (Figure 4c), i.e., the T. caerulescens plants in either side of the pot each accumulated ∼170 µg. The same was seen for T. caerulescens in the ZnS treatment; the 25.6 µg of water-extractable Zn per pot was more than accounted for by the Zn accumulated by both T. caerulescens (Figure 5c, 86 µg) and T. arvense (Figure 3c, 2 µg) when sharing a rhizosphere. Indeed, T. caerulescens accumulated more Zn than that in the water-extractable fraction; some Zn was obtained from less-available pools during growth as previously reported (8, 11). 3240
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Ecologically, this effect of T. caerulescens on the toxicity of metal-rich soils might influence the growth of surrounding species. If T. caerulescens depletes significant proportions of the soluble-Zn from its rhizosphere in its native soils, then this might protect juxtaposed roots of nonaccumulator species from extreme toxicity. Positive effects of one plant enabling the establishment of others species through the amelioration of harsh environmental factors is known to lead to such spatial associations (“nucleation” (17, 18)). This has not previously been explored for hyperaccumulator plants on highly toxic metalliferous soils. Thlaspi caerulescens can be a pioneer colonist on metalliferous river gravels and mine wastes (2, 3, 19). Could the ameliorative effects shown by the present study promote the rate of colonization of mine spoils by other species? When considering the potential effects of root interactions between species in the field however, caution must be exercised, since the reduction in soluble-Zn will only be highly localized in the rhizosphere of the hyperaccumulator. Over time, and over a greater distance, the metal content of the soil is unlikely to change, and hence the protective effects might only be small. This question deserves further investigation. Our findings have practical applications in the revegetation of metal-contaminated soils. Removing the toxic (i.e., soluble) metal fraction from soils could protect cocropped species from metal toxicity. In effect, cocropping develops further the concept of bioavailable element stripping (20), where a precropping of metal accumulating plants was proposed to phytoextract the soluble or toxic fraction of metal from the soil before planting a crop species. Here we propose that concurrently establishing a mixed-culture of hyperaccumulators with less-tolerant species may speed a revegetation operation or enhance the growth of crops on moderately contaminated soils. In this situation, the intention is to aid establishment of other species; the hyperaccumulator plants would not necessarily be harvested to remove the metals from the site as in conventional phytoextraction. We tested this idea by examining the effects of cocropping T. arvense with T. caerulescens on Zn- and Cd-contaminated tailings collected from an abandoned mine site (Table 1). The biomass of the T. arvense was increased by ∼38% when cocropped with T. caerulescens (data not shown) but not significantly so (P > 0.05). As in the artificially enriched soils, the concentrations of Zn and Cd in the shoots of T. arvense were reduced in the cocropped treatment, but these were also not significant (P > 0.05; data not shown). These results suggest that T. arvense benefited from cocropping with T. caerulescens; however, the combination of the very high metal concentrations and poor nutrient status of the tailings resulted in poor growth of both test species in that experiment. This study is the first to show the potential for a “phytoprotection” role for hyperaccumulator plants in contaminated soils. Further studies are now being conducted using moderately contaminated soils to determine whether greater benefits of companion planting of hyperaccumulators with less tolerant species can be realized by selection of ecotypes of both species that are most appropriate to the specific edaphic environment into which they are planted.
Acknowledgments We wish to thank Paul Cooke and Andrew Fairburn for their assistance with chemical analyses. This research was funded jointly by IACR-Rothamsted and the Biotechnology and Biological Sciences Research Council (BBSRC) through a CASE postgraduate studentship award to S.N.W.
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Received for review February 14, 2001. Revised manuscript received May 14, 2001. Accepted May 15, 2001. ES010644M
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