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Photosynthesis and growth responses of giant reed (Arundo donax L.) to the heavy metals Cd and Ni
Environment International 31 (2005) 243 – 249 www.elsevier.com/locate/envint
Photosynthesis and growth responses of giant reed (Arundo donax L.) to the heavy metals Cd and Ni E.G. Papazogloua,*, G.A. Karantouniasa, S.N. Vemmosb, D.L. Bouranisc a
Faculty of Natural Resources Management and Agricultural Engineering, Agricultural Hydraulics Laboratory, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece b Faculty of Crop Science and Production, Pomology Laboratory, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece c Faculty of Agricultural Biotechnology, Plant Physiology Laboratory, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece Available online 6 November 2004
Abstract Giant reed (Arundo donax L.) was grown on surface soil and irrigated with mixed heavy metal solutions of Cd(II) and Ni(II) to study the impact of these heavy metals on its growth and photosynthesis. The tested concentrations were 5, 50, and 100 ppm for each heavy metal against the control and resulted in high cadmium and nickel (DTPA extractable) concentrations in the top zone of the pot soil. The examined parameters, namely, stem height and diameter, number of nodes, fresh and dry weight of leaves, and net photosynthesis (Pn) were not affected, indicating that plants tolerate the high concentrations of Cd and Ni. As giant reed plants are very promising energy plants, they can be cultivated in contaminated soils to provide biomass for energy production purposes. D 2004 Elsevier Ltd. All rights reserved. Keywords: Arundo donax; Photosynthesis; Growth; Heavy metals; Cadmium; Nickel
1. Introduction Arundo donax L. (giant reed, Poaceae) is a potentially high-yielding nonfood crop, which can be used for the production of energy, paper pulp, and wooden building materials. It is a robust invasive perennial grass, wildgrowing in southern European regions and other Mediterranean countries (El Bassam, 1998). Giant reed can easily adapt to different ecological conditions and grow in all types of soils (Perdue, 1958; Giant reed (Arundo donax L.) Network, 2001). Heavy metal contamination of soil and water resources is a growing problem in many areas around the world. Although, at trace levels, heavy metals are natural components of soils, activities such as mining, industry,
* Corresponding author. Tel.: +30 210 5294067; fax: +30 210 5294075. E-mail address: [email protected] (E.G. Papazoglou). 0160-4120/$ - see front matter D 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2004.09.022
and localised agriculture have contributed to undesirable accumulations of toxic metals in the environment (Alloway, 1995; Kabada-Pendias and Pendias, 1992). Metal concentrations in soils range from traces to as high as 100,000 mg kg 1 (Blaylock and Huang, 2000), depending on the location and the type of metal. Among the heavy metals, cadmium is of special concern due to its potential toxicity to biota at low concentrations (Das et al., 1997). Nickel, although essential for plants at low concentrations, is, however, toxic at higher concentrations. Soils derived from ultramafic rocks typically contain 0.1% to 1.0% Ni. In many regions of Greece, such as Lavrio–Attica, there is a high level of soil contamination with multiple heavy metals, including cadmium (at 66 mg/kg) and nickel (at 73 mg/kg) (Reeves and Baker, 2000). The Lavrio peninsula is an area heavily contaminated with Cd due to the intensive mining and metallurgical activities that took place over a period of 2700 years. High Ni concentrations are found in regions different to Cd, away from mining sites, which
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Table 1 Physical and chemical characteristics of the used surface soil (20 cm depth) taken from an uncultivated land of the University campus Soil characteristics Clay (%) Silt (%) Sand (%) PH CaCO3 (%) Organic matter (%) Conductivity (mS cm 1) `) Resistance (U
40.6 24.3 35.1 7.55 36.1 3.2 2.15 470
indicate that its origin is probably due to the nature of parent material. Plants are able to employ several strategies for survival when exposed to heavy metals (Monni et al., 2001). Heavy metal resistance can be based on either avoidance or tolerance mechanisms. Plants can be protected externally against metals or they can tolerate high tissue concentrations through specific physiological mechanisms (Baker, 1987). According to Dahmani-Muller et al. (2000), a few of the higher plant species have adaptations that enable them to survive and to reproduce in soils heavily contaminated with Zn, Cu, Pb, Cd, Ni, and As. Such species are divided into two main groups: the so-called pseudometallophytes that grow on both contaminated and noncontaminated soils and the absolute metallophytes that grow only on metalcontaminated and naturally metal-rich soils. Depending on plant species, metal tolerance may result from two basic strategies: metal exclusion and metal accumulation (Baker, 1981, 1987). The exclusion strategy, comprising avoidance of metal uptake and restriction of metal transport to the shoots, is usually used by pseudometallophytes. These plants are therefore currently used to revegetate bare soil areas (e.g., in phytostabilisation technology), i.e., where the lack of vegetation results from excessively high metal concentrations. The accumulation strategy consists of strong concentration of metals in plant tissues. Hyperaccumulators are those plants with a highly abnormal level of metal accumulation in their aerial tissues (Reeves and Baker, 2000). The use of hyperaccumulators for phytoextraction relies on their ability to absorb metal contaminants from the soil and to translocate them to aerial plant parts. Usually, hyperaccumulators are plants with slow growth rates and low biomass production. Extensive research has been carried out to screen for metal hyperaccumulating plants from high biomass species (Blaylock and Huang, 2000). Over 400 plant species of hyperaccumulators from all over the world can accumulate high concentrations of metals at contaminated sites. From these, 317 taxa are nickel hyperaccumulators (Baker et al., 2000). Besides the fact that giant reed is widely spread in Lavrio–Attica (a heavy metal contaminated region, as explained above), it also (i) produces high biomass yield (dry matter up to 32–37 tons/ha) (Shatalov and Pereira, 2002) and (ii) is a highly pest-resistant crop (Lewandowski
et al., 2003), as many hyperaccumulator plants are (Baker et al., 2000). To our knowledge, no research concerned the effect of heavy metals on A. donax has been reported. Thus, our objective was to determine the feasibility of using A. donax as a means of utilizing heavy-metalcontaminated land of Lavrio-type in a productive manner (in this case, in the production of biomass fuel). For the experimental setup, contamination was achieved using an irrigation method. The application of heavy metals through irrigation was determined as being particularly effective as the achieved DTPA extractable soil concentrations of cadmium and nickel were very high.
2. Materials and methods 2.1. Experimental setup A 2-year pot experiment was set up in the experimental fields of the Agricultural University of Athens. On April of 2002, 400 rhizomes of giant reed (A. donax L.) were collected from the Kopais lake area in Viotia, Greece and planted in rooting beds filled with sand. After 30 days, on the 15th of May 2002, 60 rhizome cuttings of 0.10 m in length were selected very carefully to have uniformity, bearing one plant each, with a stem of 15F0.5 cm in height and 51 mm in diameter. All other rhizome buds were removed. Each selected rhizome was transplanted to a 0.500.50-m plastic pot. During both growing periods, one stem per pot was kept. Each pot contained 60 kg of tilled homogenized air-dried surface soil (20 cm depth) passed through a 1-cm sieve. The soil was taken from land of the University campus, which had been uncultivated for several years, and its physical and chemical characteristics are given in Table 1. An automated drip irrigation system was developed to provide plants with aqueous solutions containing cadmium and nickel in various combinations of concentrations. In the beginning of the irrigation system were placed four tanks (one for each treatment) of 500 l each, in which the heavy metal solutions were prepared. Constant flow drippers were used for irrigation. The moisture content of the pots was adjusted to 65% water-holding capacity (WHC). The added irrigation quantity was calculated every 3 days by weighing the pots. Throughout the cultivation period, the irrigation Table 2 Metal concentrations (in ppm and mM) of the three treatments, II (5 ppm), III (50 ppm), and IV (100 ppm), against the control Treatments
I (control) II III IV
Concentrations Ppm of each metal
mM Ni
Cd
0 5 50 100
0 0.085 0.85 1.7
0 0.05 0.24 0.48
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Table 3 DTPA-extractable concentrations of bioavailable Cd and Ni in soil for both experimental years Treatment
0 ppm
Zone
Top
5 ppm
Year 2002 2003 Year 2002 2003
DTPA-extractable cadmium (ppm) 0.7 0.4 0.2 0.6 0.2 0.2 DTPA extractable nickel (ppm) 2.6 2.4 2.5 1.8 2.4 2.3
Mid
Base
Top
50 ppm Mid
Base
7.0 6.4
0.8 1.1
0.5 0.4
32.3 25.3
3.5 3.0
3.0 2.7
Top
100 ppm Mid
Base
Top
Mid
Base
73.0 63.4
1.8 1.2
0.7 0.3
319.7 358.1
15.8 1.6
0.9 0.7
224.4 119.2
8.2 5.1
3.9 1.6
509.3 440.0
36.4 6.2
3.7 2.7
Pot soil was separated into three equal zones (top, mid, and base), and heavy metal concentrations were determined on samples of each zone in all four treatments.
system was thoroughly checked for correct functioning, and the quantity supplied by each dripper was tested every 20 days so as to ensure each plant received the same amount of the treatment solutions. Irrigation took place every day at 20:30 so as to minimize losses due to evaporation from the pots. A completely randomized experimental design was applied using 15 repetitions for each of the four treatments: I (control), II (5 ppm), III (50 ppm), and IV (100 ppm) (Table 2). The heavy metals were supplied as Cd(NO3)2d 4H2O and Ni(NO3)2d 6H2O. The irrigation with heavy metals for the first experimental year began on July 1, 2002 and ended on October 15,
2002. The period between May 15 and June 30 was an adaptation period for the plants. The first measurements and determinations were recorded on July 16, 2002. On the second year, the irrigation of heavy metals (on the same experimental plants) began on July 3, 2003 and ended on October 30, 2003. In the period in between (October 2002– June 2003), irrigation was applied with water only when needed.
Fig. 1. Time course of and the effect of treatments on stem height in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
Fig. 2. Time course of and the effect of treatments on stem diameter in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
2.2. Plant growth measurements The stem height and diameter and the number of nodes were recorded every 15 days during both growing periods. The appearance of the inflorescence took place mid-
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for the first year and 1700F300 Amol m 2 s 1 for the second year. Air and leaf temperatures in the chamber were recorded and were 34F2 and 33F3 8C for the first year and 35F2 and 34F3 8C for the second year, respectively. Relative humidity was about 43%F10 and 46%F20 for each of the 2 years. Net photosynthesis rates were calculated in the computing system of the IRGA based on equations of Leuning and Sands (Vemmos, 1994) and expressed as Amol CO2 m 2 s 1, respectively. 2.4. Soil analysis At the end of the experiment, pot soil was separated into three equal zones (top, mid, and base) to reveal whether a gradient of bioavailable metal contents existed with depth. A sample of each zone was analyzed to determine the bioavailable concentrations of Cd and Ni in all four treatments. The bioavailable forms of the metals were determined by extraction from soil with diethylenetriaminepentaacetic acid–CaCl2–triethanolamine (DTPA) (Lind-
Fig. 3. Time course of and the effect of treatments on number of nodes in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
September. At the end of the reproductive periods, the leaves of the plants were collected and measured for fresh weight and then dried at 70 8C for 3 days, at which their dry weight was recorded as well. 2.3. Net photosynthesis measurements Net photosynthesis (Pn) was measured in the field three times during each growing period using a closed portable infrared gas analysis (IRGA) system (LI-COR, LI-6200 model). Measurements were taken between 09:00 and 11:00 h. Net photosynthesis was measured on two or three leaves per plant according to the plant growth. On the first measurement date, Pn was recorded on the youngest upper leaf (leaf B) and on a mature leaf (leaf A: the sixth lower than leaf B), and both were marked to measure Pn again on the same leaves in the next measurements. On the second date, Pn was measured in leaves A and B and also in the new youngest upper leaf C. On the third date, the plants already had panicles, and the same marked leaves A, B, and C were measured again. A 10.88-cm2 leaf area was enclosed in a 1/4-l chamber connected to the IRGA. Airflow rate into the IRGA system was 470 Amol s 1. Photosynthetic active radiation (PAR), measured with a quantum sensor connected to the chamber, was found to be 1500F350 Amol m 2 s 1
Fig. 4. The effect of treatments on total fresh weight of the leaves in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
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These values were 4.5 and 5.7 times higher than those of the top zone of treatment III (50 ppm). Likewise, Ni concentrations on the top zone were 509 and 440 ppm for treatment IV for the years 2002 and 2003, respectively, and they were 2.3 and 3.7 times higher than those of top zone of treatment III. The decline in metal concentrations with depth was characteristically high. During the vegetative stages of plant development, all plants of all treatments showed no detrimental or toxic symptoms and increased in stem height, diameter, and number of nodes (Figs. 1–3). Those morphological characteristics, as well as fresh and dry weight of the leaves (Figs. 4, 5), did not show statistically significant differences (Scheffe test, pb0.05) between control and heavy-metaltreated plants for both years. Pn rates did not differ significantly (Scheffe test, pb0.05) between the treatments on the three dates of measurements in both years of the experiment (Figs. 6–8). The results indicate an increased Pn rate in B and C leaves as compared with A (older) leaves. There was a tendency for higher photosynthetic rates in the second year and, in particular, for B and C leaves compared to the first year. Net photosynthesis ranged between 15.3 and 25.4 Amol m 2 s 1 for the
Fig. 5. The effect of treatments on total dry weight of the leaves in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
say and Norvell, 1978), and the concentrations of Cd and Ni were quantified by atomic absorption spectrometry (AAS). 2.5. Statistical analysis Data were analysed with analysis of variance (ANOVA), and the statistical significance of the differences between treatment means were determined by means of the Scheffe test ( pb0.05).
3. Results Substantial differences in Cd and Ni concentrations were observed, both among different soil zones and among treatments (Table 3). The top zone exhibited the highest metal contents for all three zones and for all treatments. The highest Cd bioavailable concentration for both years of the experiment was up to 320 and 358 ppm, respectively, and was measured in the top zone of treatment IV (100 ppm).
Fig. 6. The effect of the treatments on net photosynthesis of A leaves (see Materials and methods) in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
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A. donax is a grass with C3 photosynthetic pathway unlike other grasses (e.g., switchgrass and miscanthus) with C4 pathway (Lewandowski et al., 2003). Physiological processes, such as photosynthesis and water status, are sensitive to heavy metals (Monni et al., 2001) in several plant species. Heavy metals have been found to inhibit electron transport in photosynthetic systems (Becerril et al., 1989) and the regenerative phase of the Calvin cycle (Weigel, 1985). Photosynthetic rates of A. donax were unaffected by the treatments, indicating that the photosynthetic system was not harmed and showed a strong tolerance of this plant to the increased heavy metal concentrations in the soil. The mean values of giant reed Pn rates found in this study were higher than those usual for C3 plants (18–20 Amol CO2 m 2 s 1; Mohr and Schopfer, 1995). Rossa et al. (1998), in a comparative study on photosynthesis of five C3 and three C4 grasses, found that A. donax had high Pn rates, higher than the other grasses (37.0 Amol CO2 m 2 s 1) under similar environmental conditions. Pn rates of our study (received under comparable con-
Fig. 7. The effect of the treatments on net photosynthesis of B leaves (see Materials and methods) in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
first year and 18.7 and 34.0 Amol m year.
2
s
1
for the second
4. Discussion The applied irrigation method resulted in a significant addition of heavy metals of very high level at the top zone of the experimental soil volume (Table 3). In spite of this, the heavy-metal-treated giant reed plants did not show any overt symptoms of toxicity, even at high-level treatment IV (100 ppm). All plants of our experiment were well developed and healthy, and it was not possible to distinguish control plants from treated plants. Furthermore, there were no statistically significant differences among all studied morphological parameters and the fresh and dry weight of control plants and the plants that grew in the presence of the mixed heavy metals. Comparing the data between the two growing periods, it is evident that a reduction in growth of all plants has occurred during the second year. This reduction was reported in control plants as well possibly due to the fact that the plants were grown in pots and not in the field.
Fig. 8. The effect of the treatments on net photosynthesis of C leaves (see Materials and methods) in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
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ditions) are in accordance with these. The same authors suggest that the high Pn rates of A. donax are attributed to the higher level of irradiance for saturation of electron transport through PSII and less photoinhibition compared to other grasses. The high photosynthetic activity of giant reed indicates a higher biomass production of this plant compared to other similar grasses with lower photosynthetic capacity.
5. Conclusions Irrigation of A. donax crop with tap water containing increased concentrations of cadmium and nickel resulted in high content of these metals in the top soil of the pots. In spite of the high heavy metal concentrations, plants did not show any detrimental effect on net photosynthesis and plant growth. It is obvious that A. donax is a plant that tolerates increased concentrations of cadmium and nickel in its rhizosphere, and thus it can be cultivated in contaminated areas for energy production purposes.
References Alloway BJ. Soil processes and the behaviour of heavy metals. In: Alloway BJ, editor. Heavy metals in soils. London7 Chapman & Hill; 1995. Baker AJM. Accumulators and excluders: strategies in the response of plants to heavy metals. J Plant Nutr 1981;3:643. Baker AJM. Metal tolerance. New Phytol 1987;106:93 – 111. Baker AJM, McGrath SP, Reeves RD, Smith JAC. Metal hyperaccumulator plants: a review of the ecology and physiology of a biological resource for phytoremediation of metal-polluted soils. In: Terry N, Banuelos G, editors. Phytoremediation of contaminated soil and water. USA7 Lewis Publishers; 2000. Becerril JM, Munoz-Rueda A, Aparicio-Tejo P, Gonzalez-Murua C. The effects of cadmium and lead on photosynthetic electron transport in clover and lucerne. Plant Physiol Biochem 1989;26:913 – 8.
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Blaylock JM, Huang JW. Phytoextraction of metals. In: Raskin I, Ensley BD, editors. Phytoremediation of toxic metals: using plants to clean up the environment. New York7 John Wiley and Sons; 2000. Dahmani-Muller H, van Oort F, Gelie B, Balabane M. Strategies of heavy metal uptake by three plant species growing near a metal smelter. Environ Pollut 2000;109:231 – 8. Das P, Samantaray S, Rout GR. Studies on cadmium toxicity in plants: a review. Environ Pollut 1997;98:29 – 36. El Bassam N. Energy plant species. Their use and impact on environment and development. London7 James & James; 1998. Kabada-Pendias A, Pendias H. Trace elements in soils and plants. . USA7 CRC Press; 1992. Giant reed (Arundo donax L.) Network, 2001. Improvement, productivity and biomass quality. (Contract No: FAIR3 CT96 2028); EU. Lewandowski I, Scurlock JMO, Lindvall E, Christou M. The development and current status of perennial rhizomatous grasses as energy crops in the US and Europe. Biomass Bioenergy 2003;25:335 – 61. Lindsay WL, Norvell WA. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci Soc Am J 1978;42:421 – 8. Mohr H, Schopfer P. Plant physiology C4 and CAM plants. Berlin7 Springer-Verlag; 1995. Monni S, Uhlig C, Junttila O, Hansen E, Hynynen J. Chemical composition and ecophysiological responses of Empetrum nigrum to aboveground element application. Environ Pollut 2001;112:417 – 26. Perdue RE. Arundo donax source of musical reeds and industrial cellulose. Econ Bot 1958;12:368 – 404. Reeves RD, Baker M. Metal-accumulating plants. In: Raskin I, Ensley BD, editors. Phytoremediation of toxic metals: using plants to clean up the environment. New York7 John Wiley and Sons; 2000. Rossa B, Tuffers AV, Naidoo G, von Willert DJ. Arundo donax L (Poaceae)—A C3 species with unusually high photosynthetic capacity. Bot Acta 1998;111:216 – 21. Shatalov A, Pereira H. Influence of stem morphology on pulp and paper properties of Arundo donax L reed. Ind Crops Prod 2002;15:77 – 83. Vemmos SN. Net photosynthesis, stomatal conductance, chlorophyll content and specific leaf weight of pistachio trees (cv Aegenes) as influenced by fruiting. J Hortic Sci 1994;69:775 – 82. Weigel HJ. The effect of Cd2+ on photosynthetic reactions of mesophyll protoplasts. Physiol Plant 1985;63:192 – 200.
Photosynthesis and growth responses of giant reed (Arundo donax L.) to the heavy metals Cd and Ni E.G. Papazogloua,*, G.A. Karantouniasa, S.N. Vemmosb, D.L. Bouranisc a
Faculty of Natural Resources Management and Agricultural Engineering, Agricultural Hydraulics Laboratory, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece b Faculty of Crop Science and Production, Pomology Laboratory, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece c Faculty of Agricultural Biotechnology, Plant Physiology Laboratory, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece Available online 6 November 2004
Abstract Giant reed (Arundo donax L.) was grown on surface soil and irrigated with mixed heavy metal solutions of Cd(II) and Ni(II) to study the impact of these heavy metals on its growth and photosynthesis. The tested concentrations were 5, 50, and 100 ppm for each heavy metal against the control and resulted in high cadmium and nickel (DTPA extractable) concentrations in the top zone of the pot soil. The examined parameters, namely, stem height and diameter, number of nodes, fresh and dry weight of leaves, and net photosynthesis (Pn) were not affected, indicating that plants tolerate the high concentrations of Cd and Ni. As giant reed plants are very promising energy plants, they can be cultivated in contaminated soils to provide biomass for energy production purposes. D 2004 Elsevier Ltd. All rights reserved. Keywords: Arundo donax; Photosynthesis; Growth; Heavy metals; Cadmium; Nickel
1. Introduction Arundo donax L. (giant reed, Poaceae) is a potentially high-yielding nonfood crop, which can be used for the production of energy, paper pulp, and wooden building materials. It is a robust invasive perennial grass, wildgrowing in southern European regions and other Mediterranean countries (El Bassam, 1998). Giant reed can easily adapt to different ecological conditions and grow in all types of soils (Perdue, 1958; Giant reed (Arundo donax L.) Network, 2001). Heavy metal contamination of soil and water resources is a growing problem in many areas around the world. Although, at trace levels, heavy metals are natural components of soils, activities such as mining, industry,
* Corresponding author. Tel.: +30 210 5294067; fax: +30 210 5294075. E-mail address: [email protected] (E.G. Papazoglou). 0160-4120/$ - see front matter D 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2004.09.022
and localised agriculture have contributed to undesirable accumulations of toxic metals in the environment (Alloway, 1995; Kabada-Pendias and Pendias, 1992). Metal concentrations in soils range from traces to as high as 100,000 mg kg 1 (Blaylock and Huang, 2000), depending on the location and the type of metal. Among the heavy metals, cadmium is of special concern due to its potential toxicity to biota at low concentrations (Das et al., 1997). Nickel, although essential for plants at low concentrations, is, however, toxic at higher concentrations. Soils derived from ultramafic rocks typically contain 0.1% to 1.0% Ni. In many regions of Greece, such as Lavrio–Attica, there is a high level of soil contamination with multiple heavy metals, including cadmium (at 66 mg/kg) and nickel (at 73 mg/kg) (Reeves and Baker, 2000). The Lavrio peninsula is an area heavily contaminated with Cd due to the intensive mining and metallurgical activities that took place over a period of 2700 years. High Ni concentrations are found in regions different to Cd, away from mining sites, which
244
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Table 1 Physical and chemical characteristics of the used surface soil (20 cm depth) taken from an uncultivated land of the University campus Soil characteristics Clay (%) Silt (%) Sand (%) PH CaCO3 (%) Organic matter (%) Conductivity (mS cm 1) `) Resistance (U
40.6 24.3 35.1 7.55 36.1 3.2 2.15 470
indicate that its origin is probably due to the nature of parent material. Plants are able to employ several strategies for survival when exposed to heavy metals (Monni et al., 2001). Heavy metal resistance can be based on either avoidance or tolerance mechanisms. Plants can be protected externally against metals or they can tolerate high tissue concentrations through specific physiological mechanisms (Baker, 1987). According to Dahmani-Muller et al. (2000), a few of the higher plant species have adaptations that enable them to survive and to reproduce in soils heavily contaminated with Zn, Cu, Pb, Cd, Ni, and As. Such species are divided into two main groups: the so-called pseudometallophytes that grow on both contaminated and noncontaminated soils and the absolute metallophytes that grow only on metalcontaminated and naturally metal-rich soils. Depending on plant species, metal tolerance may result from two basic strategies: metal exclusion and metal accumulation (Baker, 1981, 1987). The exclusion strategy, comprising avoidance of metal uptake and restriction of metal transport to the shoots, is usually used by pseudometallophytes. These plants are therefore currently used to revegetate bare soil areas (e.g., in phytostabilisation technology), i.e., where the lack of vegetation results from excessively high metal concentrations. The accumulation strategy consists of strong concentration of metals in plant tissues. Hyperaccumulators are those plants with a highly abnormal level of metal accumulation in their aerial tissues (Reeves and Baker, 2000). The use of hyperaccumulators for phytoextraction relies on their ability to absorb metal contaminants from the soil and to translocate them to aerial plant parts. Usually, hyperaccumulators are plants with slow growth rates and low biomass production. Extensive research has been carried out to screen for metal hyperaccumulating plants from high biomass species (Blaylock and Huang, 2000). Over 400 plant species of hyperaccumulators from all over the world can accumulate high concentrations of metals at contaminated sites. From these, 317 taxa are nickel hyperaccumulators (Baker et al., 2000). Besides the fact that giant reed is widely spread in Lavrio–Attica (a heavy metal contaminated region, as explained above), it also (i) produces high biomass yield (dry matter up to 32–37 tons/ha) (Shatalov and Pereira, 2002) and (ii) is a highly pest-resistant crop (Lewandowski
et al., 2003), as many hyperaccumulator plants are (Baker et al., 2000). To our knowledge, no research concerned the effect of heavy metals on A. donax has been reported. Thus, our objective was to determine the feasibility of using A. donax as a means of utilizing heavy-metalcontaminated land of Lavrio-type in a productive manner (in this case, in the production of biomass fuel). For the experimental setup, contamination was achieved using an irrigation method. The application of heavy metals through irrigation was determined as being particularly effective as the achieved DTPA extractable soil concentrations of cadmium and nickel were very high.
2. Materials and methods 2.1. Experimental setup A 2-year pot experiment was set up in the experimental fields of the Agricultural University of Athens. On April of 2002, 400 rhizomes of giant reed (A. donax L.) were collected from the Kopais lake area in Viotia, Greece and planted in rooting beds filled with sand. After 30 days, on the 15th of May 2002, 60 rhizome cuttings of 0.10 m in length were selected very carefully to have uniformity, bearing one plant each, with a stem of 15F0.5 cm in height and 51 mm in diameter. All other rhizome buds were removed. Each selected rhizome was transplanted to a 0.500.50-m plastic pot. During both growing periods, one stem per pot was kept. Each pot contained 60 kg of tilled homogenized air-dried surface soil (20 cm depth) passed through a 1-cm sieve. The soil was taken from land of the University campus, which had been uncultivated for several years, and its physical and chemical characteristics are given in Table 1. An automated drip irrigation system was developed to provide plants with aqueous solutions containing cadmium and nickel in various combinations of concentrations. In the beginning of the irrigation system were placed four tanks (one for each treatment) of 500 l each, in which the heavy metal solutions were prepared. Constant flow drippers were used for irrigation. The moisture content of the pots was adjusted to 65% water-holding capacity (WHC). The added irrigation quantity was calculated every 3 days by weighing the pots. Throughout the cultivation period, the irrigation Table 2 Metal concentrations (in ppm and mM) of the three treatments, II (5 ppm), III (50 ppm), and IV (100 ppm), against the control Treatments
I (control) II III IV
Concentrations Ppm of each metal
mM Ni
Cd
0 5 50 100
0 0.085 0.85 1.7
0 0.05 0.24 0.48
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Table 3 DTPA-extractable concentrations of bioavailable Cd and Ni in soil for both experimental years Treatment
0 ppm
Zone
Top
5 ppm
Year 2002 2003 Year 2002 2003
DTPA-extractable cadmium (ppm) 0.7 0.4 0.2 0.6 0.2 0.2 DTPA extractable nickel (ppm) 2.6 2.4 2.5 1.8 2.4 2.3
Mid
Base
Top
50 ppm Mid
Base
7.0 6.4
0.8 1.1
0.5 0.4
32.3 25.3
3.5 3.0
3.0 2.7
Top
100 ppm Mid
Base
Top
Mid
Base
73.0 63.4
1.8 1.2
0.7 0.3
319.7 358.1
15.8 1.6
0.9 0.7
224.4 119.2
8.2 5.1
3.9 1.6
509.3 440.0
36.4 6.2
3.7 2.7
Pot soil was separated into three equal zones (top, mid, and base), and heavy metal concentrations were determined on samples of each zone in all four treatments.
system was thoroughly checked for correct functioning, and the quantity supplied by each dripper was tested every 20 days so as to ensure each plant received the same amount of the treatment solutions. Irrigation took place every day at 20:30 so as to minimize losses due to evaporation from the pots. A completely randomized experimental design was applied using 15 repetitions for each of the four treatments: I (control), II (5 ppm), III (50 ppm), and IV (100 ppm) (Table 2). The heavy metals were supplied as Cd(NO3)2d 4H2O and Ni(NO3)2d 6H2O. The irrigation with heavy metals for the first experimental year began on July 1, 2002 and ended on October 15,
2002. The period between May 15 and June 30 was an adaptation period for the plants. The first measurements and determinations were recorded on July 16, 2002. On the second year, the irrigation of heavy metals (on the same experimental plants) began on July 3, 2003 and ended on October 30, 2003. In the period in between (October 2002– June 2003), irrigation was applied with water only when needed.
Fig. 1. Time course of and the effect of treatments on stem height in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
Fig. 2. Time course of and the effect of treatments on stem diameter in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
2.2. Plant growth measurements The stem height and diameter and the number of nodes were recorded every 15 days during both growing periods. The appearance of the inflorescence took place mid-
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for the first year and 1700F300 Amol m 2 s 1 for the second year. Air and leaf temperatures in the chamber were recorded and were 34F2 and 33F3 8C for the first year and 35F2 and 34F3 8C for the second year, respectively. Relative humidity was about 43%F10 and 46%F20 for each of the 2 years. Net photosynthesis rates were calculated in the computing system of the IRGA based on equations of Leuning and Sands (Vemmos, 1994) and expressed as Amol CO2 m 2 s 1, respectively. 2.4. Soil analysis At the end of the experiment, pot soil was separated into three equal zones (top, mid, and base) to reveal whether a gradient of bioavailable metal contents existed with depth. A sample of each zone was analyzed to determine the bioavailable concentrations of Cd and Ni in all four treatments. The bioavailable forms of the metals were determined by extraction from soil with diethylenetriaminepentaacetic acid–CaCl2–triethanolamine (DTPA) (Lind-
Fig. 3. Time course of and the effect of treatments on number of nodes in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
September. At the end of the reproductive periods, the leaves of the plants were collected and measured for fresh weight and then dried at 70 8C for 3 days, at which their dry weight was recorded as well. 2.3. Net photosynthesis measurements Net photosynthesis (Pn) was measured in the field three times during each growing period using a closed portable infrared gas analysis (IRGA) system (LI-COR, LI-6200 model). Measurements were taken between 09:00 and 11:00 h. Net photosynthesis was measured on two or three leaves per plant according to the plant growth. On the first measurement date, Pn was recorded on the youngest upper leaf (leaf B) and on a mature leaf (leaf A: the sixth lower than leaf B), and both were marked to measure Pn again on the same leaves in the next measurements. On the second date, Pn was measured in leaves A and B and also in the new youngest upper leaf C. On the third date, the plants already had panicles, and the same marked leaves A, B, and C were measured again. A 10.88-cm2 leaf area was enclosed in a 1/4-l chamber connected to the IRGA. Airflow rate into the IRGA system was 470 Amol s 1. Photosynthetic active radiation (PAR), measured with a quantum sensor connected to the chamber, was found to be 1500F350 Amol m 2 s 1
Fig. 4. The effect of treatments on total fresh weight of the leaves in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
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These values were 4.5 and 5.7 times higher than those of the top zone of treatment III (50 ppm). Likewise, Ni concentrations on the top zone were 509 and 440 ppm for treatment IV for the years 2002 and 2003, respectively, and they were 2.3 and 3.7 times higher than those of top zone of treatment III. The decline in metal concentrations with depth was characteristically high. During the vegetative stages of plant development, all plants of all treatments showed no detrimental or toxic symptoms and increased in stem height, diameter, and number of nodes (Figs. 1–3). Those morphological characteristics, as well as fresh and dry weight of the leaves (Figs. 4, 5), did not show statistically significant differences (Scheffe test, pb0.05) between control and heavy-metaltreated plants for both years. Pn rates did not differ significantly (Scheffe test, pb0.05) between the treatments on the three dates of measurements in both years of the experiment (Figs. 6–8). The results indicate an increased Pn rate in B and C leaves as compared with A (older) leaves. There was a tendency for higher photosynthetic rates in the second year and, in particular, for B and C leaves compared to the first year. Net photosynthesis ranged between 15.3 and 25.4 Amol m 2 s 1 for the
Fig. 5. The effect of treatments on total dry weight of the leaves in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
say and Norvell, 1978), and the concentrations of Cd and Ni were quantified by atomic absorption spectrometry (AAS). 2.5. Statistical analysis Data were analysed with analysis of variance (ANOVA), and the statistical significance of the differences between treatment means were determined by means of the Scheffe test ( pb0.05).
3. Results Substantial differences in Cd and Ni concentrations were observed, both among different soil zones and among treatments (Table 3). The top zone exhibited the highest metal contents for all three zones and for all treatments. The highest Cd bioavailable concentration for both years of the experiment was up to 320 and 358 ppm, respectively, and was measured in the top zone of treatment IV (100 ppm).
Fig. 6. The effect of the treatments on net photosynthesis of A leaves (see Materials and methods) in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
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A. donax is a grass with C3 photosynthetic pathway unlike other grasses (e.g., switchgrass and miscanthus) with C4 pathway (Lewandowski et al., 2003). Physiological processes, such as photosynthesis and water status, are sensitive to heavy metals (Monni et al., 2001) in several plant species. Heavy metals have been found to inhibit electron transport in photosynthetic systems (Becerril et al., 1989) and the regenerative phase of the Calvin cycle (Weigel, 1985). Photosynthetic rates of A. donax were unaffected by the treatments, indicating that the photosynthetic system was not harmed and showed a strong tolerance of this plant to the increased heavy metal concentrations in the soil. The mean values of giant reed Pn rates found in this study were higher than those usual for C3 plants (18–20 Amol CO2 m 2 s 1; Mohr and Schopfer, 1995). Rossa et al. (1998), in a comparative study on photosynthesis of five C3 and three C4 grasses, found that A. donax had high Pn rates, higher than the other grasses (37.0 Amol CO2 m 2 s 1) under similar environmental conditions. Pn rates of our study (received under comparable con-
Fig. 7. The effect of the treatments on net photosynthesis of B leaves (see Materials and methods) in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
first year and 18.7 and 34.0 Amol m year.
2
s
1
for the second
4. Discussion The applied irrigation method resulted in a significant addition of heavy metals of very high level at the top zone of the experimental soil volume (Table 3). In spite of this, the heavy-metal-treated giant reed plants did not show any overt symptoms of toxicity, even at high-level treatment IV (100 ppm). All plants of our experiment were well developed and healthy, and it was not possible to distinguish control plants from treated plants. Furthermore, there were no statistically significant differences among all studied morphological parameters and the fresh and dry weight of control plants and the plants that grew in the presence of the mixed heavy metals. Comparing the data between the two growing periods, it is evident that a reduction in growth of all plants has occurred during the second year. This reduction was reported in control plants as well possibly due to the fact that the plants were grown in pots and not in the field.
Fig. 8. The effect of the treatments on net photosynthesis of C leaves (see Materials and methods) in each of the years 2002 and 2003. A Scheffe test ( pb0.05) was performed, and no statistical significant differences were found between the examined treatments. Error bars represent the experimental minimum and maximum values to establish a range for diagnostic purposes.
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ditions) are in accordance with these. The same authors suggest that the high Pn rates of A. donax are attributed to the higher level of irradiance for saturation of electron transport through PSII and less photoinhibition compared to other grasses. The high photosynthetic activity of giant reed indicates a higher biomass production of this plant compared to other similar grasses with lower photosynthetic capacity.
5. Conclusions Irrigation of A. donax crop with tap water containing increased concentrations of cadmium and nickel resulted in high content of these metals in the top soil of the pots. In spite of the high heavy metal concentrations, plants did not show any detrimental effect on net photosynthesis and plant growth. It is obvious that A. donax is a plant that tolerates increased concentrations of cadmium and nickel in its rhizosphere, and thus it can be cultivated in contaminated areas for energy production purposes.
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