- Email: [email protected]
The Effect of pH and Plaque on the Uptake of Cu and Mn in Phragmites australis(Cav.) Trin ex. Steudel
Annals of Botany 86: 647±653, 2000 doi:10.1006/anbo.2000.1191, available online at http://www.idealibrary.com on
The Eect of pH and Plaque on the Uptake of Cu and Mn in Phragmites australis (Cav.) Trin ex. Steudel L . C . B AT T Y * {, A . J. M . B A K E R {, B . D. W H E E L E R{ and C . D . C U R T I S{ {Department of Plant and Animal Sciences, University of Sheeld, Sheeld S10 2TN, UK and {Department of Earth Sciences, University of Manchester, Manchester M13 9PL, UK Received: 10 August 1999 Returned for revision: 3 January 2000
Accepted: 24 March 2000
The physico-chemical properties of iron oxyhydroxide plaques formed on the roots of Phragmites australis under ®eld and laboratory conditions were determined using electron microscopy and energy dispersive spectrometry (EDS) analysis. Plaques were present as an amorphous coating on roots with an uneven distribution. They were shown to be composed of iron in both the ®eld and laboratory, and phosphorus was found to be adsorbed onto the surface of plaques formed in the laboratory. The uptake of copper and manganese in the presence and absence of plaque was investigated under two dierent pH regimes. Concentrations of Cu were lower in the shoots of P. australis in the presence of plaque (565 mg kg ÿ1) than when it was absent (1400 mg kg ÿ1), under growth conditions of higher pH (6.0). The adsorption of Cu and Mn onto the plaque surface was not the mechanism by which plaque reduced the uptake of other metals. Alternatively, the plaque may simply act as a physical barrier. Under conditions of lower pH (3.5) the activity of hydrogen ions at the root surface interfered with the movement of metals into the root and # 2000 Annals of Botany Company masked any potential eect of iron plaque. Key words: Phragmites australis, iron oxyhydroxide plaque, pH, manganese, copper, EDS, SEM, wetlands.
I N T RO D U C T I O N Acid mine drainage is a major source of metal contamination in many regions of the world. Discharges are characterized by low pH (53.5), elevated levels of metals such as Cu, Mn, Fe and Zn, together with low levels of organic compounds. For a number of years, wetlands, both natural and constructed, have been used to remove potentially toxic metals from contaminated drainage (Eger and Lapakko, 1989; Wildeman and Laudon, 1989; Lan et al., 1992; Dodds-Smith et al., 1995; Beining and Otte, 1996; Scholes et al., 1998). Wetland plant species, including Phragmites australis and Typha latifolia, are an important constituent of these passive treatments, but the strategy by which these species survive in highly metalliferous sites remains unclear. Coatings of iron (oxyhydr-)oxides known as `iron plaques' have been observed on a number of wetland plant species, including those growing in contaminated areas (Greipsson, 1994; Snowden and Wheeler, 1995; Sundby et al., 1998; Ye et al., 1998; Wang and Peverly, 1999). The exact cause of iron precipitation around roots is unknown but the oxidizing activity of roots, together with the action of micro-organisms, is thought to be involved. Iron oxide deposits are generally seen as an irregular porous coating on the root surface (Bacha and Hossner, 1977; St Cyr et al., 1993). Plaque deposits have been analysed using a variety of techniques including X-ray diraction and energy dispersive X-ray microanalysis. The coatings have been reported to consist of lepidocrocite * For correspondence. Fax 0191 222 6669, e-mail [email protected]
0305-7364/00/090647+07 $35.00/00
(g-FeOOH; Bacha and Hossner, 1977), a mixture of lepidocrocite and geothite (a-FeOOH; Chen et al., 1980a; St Cyr et al., 1993) and ferric phosphate (Snowden and Wheeler, 1995). Root plaques may also contain a variety of metals and metalloids which include aluminium, arsenic, cadmium, chromium, mercury, nickel, lead and zinc (Otte et al., 1987, 1989, 1995; St Cyr and Crowder, 1987, 1990; Greipsson and Crowder, 1992; St Cyr and Campbell, 1996; Doyle and Otte, 1997; Ye et al., 1997a, 1998; Sundby et al., 1998). The formation of iron plaques around the roots of wetland plants may constitute an adaptation to stressed environments. In ¯ooded areas the chemical reduction of iron and manganese compounds results in the accumulation of high levels of these bioavailable elements. In addition, there may be potentially phytotoxic levels of metals or metalloids, particularly in acid soils. Iron oxides present in soils and sediments have high speci®c surface areas and possess -OH functional groups which are capable of reacting with metals and other cations and anions (Kuo, 1986). It is possible that iron hydroxides forming on the roots of wetland plants have similar properties and may therefore immobilize and prevent the uptake of phytotoxic metals. This `exclusion' hypothesis is supported by reports of the amelioration of the toxic eects of phytotoxic metals in a variety of species. It has been reported that the presence of iron plaque on Oryza sativa seedlings improved growth under mildly toxic conditions of copper and (or) nickel exposure (Greipsson and Crowder, 1992; Greipsson, 1994). However, iron plaque also appeared to enhance the uptake of iron and it was proposed that the high concentration of # 2000 Annals of Botany Company
648
Batty et al.ÐMetal Uptake in Phragmites australis
iron in the leaves enabled competition by iron with copper for sensitive metabolic sites (Greipsson, 1994). Crowder et al. (1987) also found that the growth of Oryza sativa exposed to 0.5 ppm copper was greater in the presence of plaque. In contrast, investigations into the growth of Typha latifolia demonstrated that the presence of plaque on the roots of seedlings did not enhance metal tolerance or growth when subjected to copper and (or) nickel, zinc and (or) lead and cadmium (Ye, 1995; Ye et al., 1997b, 1998). However, the presence of plaque did reduce root length. Extensive research has also been undertaken to determine whether the presence of plaque reduces the uptake of phytotoxic metals. Iron plaques have been shown to act as a ®lter for the movement of iron, copper, zinc, nickel and cadmium into rhizomes and shoots (Greipsson and Crowder, 1992; Greipsson, 1994; Wang and Peverly, 1996). Otte et al. (1987, 1989) also found that a heavy coating of iron plaque may act as a barrier to zinc uptake in Aster tripolium L., but when the coating was light, zinc uptake was enhanced. The majority of research has shown that the formation of plaque does not impede the uptake of toxic metals. This has been demonstrated for the uptake of iron in Oryza sativa (Benckiser et al., 1984), manganese in Picea mariana Mill, Pinus resinosa Ait. (Levan and Riha, 1986) and Oryza sativa (Crowder and Coltman, 1993), copper and zinc in Phragmites australis (St Cyr and Crowder, 1987), copper and nickel in Typha latifolia (Ye et al., 1997a) and zinc and lead in Typha latifolia (Ye et al., 1998). Information in the literature is con¯icting, and the precise role of plaque remains unresolved. The research that has been undertaken has included investigation of seedlings from a wide variety of species including freshwater, saltwater and non-wetland plants, together with the use of material from both the ®eld and the laboratory. In the controlled conditions of the laboratory a number of techniques have been used with diering growth media (including nutrient solutions, sand and agar), a variety of pH conditions, diering metal sources (which has already been proved to be an important factor; Taylor et al., 1984), together with a number of dierent analytical techniques. It is therefore dicult to compare dierent investigations and to extract any de®nitive conclusions from the literature. The aim of this study was to determine the chemical and structural characteristics of iron plaques formed both in the ®eld and laboratory in order to assess the application of controlled laboratory experiments to the ®eld situation. In addition, an investigation was undertaken into the uptake of metals by the wetland species Phragmites australis in the presence and absence of plaque, in both acidic and more neutral pH conditions. The majority of investigations into the eects of plaque have been conducted in a narrow pH range between 5 and 7 (Mac®e and Crowder, 1987; St Cyr and Crowder, 1989); however wetland plants are frequently used to remediate acid mine drainage and therefore experiments need to be constructed in both acidic and neutral environments. A number of analytical techniques were used in order to achieve a more comprehensive set of results that could be compared.
M AT E R I A L S A ND M E T H O D S Seeds of Phragmites australis Trin. ex Steudel were collected from a non-contaminated site at Felixstowe, UK. The seeds were germinated at 218C and seedlings were grown supported on alkathene beads1 for 14 d in 10 % Rorison's solution (Hewitt, 1966). Seedlings of a uniform size were selected and ten were transplanted into blackened Perspex vessels (28 17 9 cm) containing 1.5 l of 10 % Rorison's solution ( pH 6.0). In total there were 36 units allowing for three replicates of all treatments, arranged in a randomized block design. The seedlings were grown for a further 63 d, the Rorison's solution being changed every 3 d. At the end of this period the pH of the solution in 18 of the containers was reduced gradually to 3.5 using 0.1 M H2SO4 . Each set of units at pH 3.5 and 6.0 were split into two subsets consisting of nine containers. In one of these subsets plaque formation was induced and in the remaining sets the roots were left clean. Plaque formation was induced on the roots of seedlings prior to metal treatment through the addition of 50 mg l ÿ1 of Fe ((NH4)2Fe(SO4)2 .6H2O) for 7 d in nutrient solution which did not contain potassium phosphate thereby preventing the interaction of Fe with P. In addition, the seedlings were placed in distilled water of the appropriate pH for 12 h before plaquing to prevent a similar interference of P. Plaque was visible on roots as an orange±brown deposit after 7 d. All seedlings were grown for a further 5 d on 10 % Rorison's solution prior to metal treatment. The two metals added were Cu (CuSO4 .5H2O) and Mn (MnSO4 .4H2O), both at a concentration of 0.5 mg l ÿ1. These were added to the appropriate containers to give the following conditions: (1) no plaque, Cu, pH 3.5; (2) no plaque, Cu, pH 6.0; (3) plaque, Cu, pH 3.5; (4) plaque, Cu, pH 6.0; (5) no plaque, Mn, pH 3.5; (6) no plaque, Mn, pH 6.0; (7) plaque, Mn, pH 3.5; and (8) plaque, Mn, pH 6.0. Control treatments contained no additional metals. During metal exposure, solutions were changed every 3 d. Roots were re-plaqued at 21 d and 50 d from the start of metal treatment to ensure that coverage of roots with plaque was maintained. Prior to the repeated exposure of roots to iron, the seedlings were placed in distilled water for 6 h before exposure to 50 mg l ÿ1 Fe for 3 d. At the end of the experiment (130 d) all plants were harvested, divided into roots and shoots and rinsed thoroughly in distilled water to remove dust contaminating the surface. A small subsample of root from each treatment was removed, freezedried and coated with carbon for scanning electron microscopy (SEM) analysis. The iron coating on the roots was removed using the dithionite±citrate±carbonate (DCB) method of Jackson (1958) as modi®ed by Taylor and Crowder (1983). Following DCB extraction, roots and shoots were dried at 408C and acid digested in 5 ml of 30 % HNO3 at 908C for a minimum of 8 h. The concentrations of Fe, Cu and Mn in the digests were determined by atomic absorption spectrophotometry (AAS). Results are presented for metal concentration in aerial parts (shoots), iron plaque (DCB extract), roots after plaque is removed (roots) and roots with plaque (whole roots). Whole root
Batty et al.ÐMetal Uptake in Phragmites australis
649
data were calculated from the root and DCB extract concentrations allowing for dierences in mass. Samples of P. australis were collected from a constructed reed bed receiving contaminated drainage from a derelict coal mine at Woolley Colliery, near Barnsley, UK. Root sections from these samples were prepared as outlined previously. Data on metal concentrations were analysed using a combination of t-tests and 2-way ANOVA followed by a Tukey test. Where necessary, data transformations were carried out, either loge or log10 . R E S U LT S Throughout the experiment all plants showed similar growth and development of both roots and shoots. Laboratory plaques Morphology. The plaque deposits were clearly visible as orange±brown coatings on the root surface, extending from 1 cm behind the growing tip along the entire length of the root. Under the SEM these deposits could be distinguished by textural dierences from the unplaqued root. Figure 1A shows a section of the plaqued root surface when grown without exposure to Mn or Cu. It is evident that plaque was present as a particulate deposit that follows the contours of cells. The coating was not uniform and plaque deposits were absent from large areas of the root surface. Chen et al. (1980b) reported the presence of cell casts and polyhedra in plaque deposits, but these were not found on roots in the present study. No evidence of penetration of the plaque into the epidermal cells was found when transverse sections of the root were studied. In the absence of iron and the presence of Mn there was no formation of manganese plaques. Chemical composition. In clean areas of the root, X-ray analysis revealed the presence of K, S and lesser amounts of Ca, P and Cl which is a residual signature of the nutrient solution. The precipitates formed in the presence of Fe had a very speci®c chemical signature and were composed of Fe and P, together with lesser amounts of S and K (Fig. 2A). It is likely that the plaques were composed of iron phosphate. In the presence of Cu or Mn the plaques had the same chemical signature, but there was no evidence of either Cu or Mn within the deposits. Field plaques Morphology. In equivalent root sections of P. australis collected from the ®eld, plaque was also present as an orange±brown deposit on the root surface, extending from 1 cm behind the root tip along the entire length of the root. Under SEM it was evident that these particulate deposits had a similar form to those found on laboratory samples (Fig. 1B). The plaque deposits were present as amorphous particulate coverings of the root surface but were not ubiquitous, and plaque was not evident in some areas. Cell
F I G . 1. Micrographs of P. australis roots grown in a controlled laboratory (A) (500), and ®eld conditions (B) (600). IP, Iron plaque; C, clean, unplaqued areas.
casts and polyhedra were not present and there was no evidence of penetration into epidermal cells. Chemical composition. The chemistry of the plaque deposits from the ®eld diered from that of the laboratory samples. The iron precipitates present were composed mainly of iron and silica with accessory elements including Al and Mn (Fig. 2B). The absence of phosphorus implied that the plaques were not composed of iron phosphates but that iron was forming a dierent compound, yet to be identi®ed. In some regions of the root surface, the plaques had a similar chemistry, but Mn was also present in greater quantities than iron. Copper was present, associated with the iron plaque in isolated areas of the root. The eect of pH and plaque on seedlings exposed to 0.5 mg l ÿ1 Mn No signi®cant eects of pH or plaque were found for root or shoot contents (Table 1). However, at higher pH a signi®cantly greater concentration of Mn was found in the DCB extract when plaque was absent than when it was present (Fig. 3A). This was also true for the whole root data. When plaque was absent there was also more Mn in the DCB and whole root extracts at pH 6.0 (Fig. 3B).
650
Batty et al.ÐMetal Uptake in Phragmites australis
T A B L E 1. Signi®cance levels of pH and plaque treatments according to two-way analysis of variance for dierent plant fractions shown in Fig. 3
Mn
Plaque
Interaction
Shoot Root DCB Whole root
50.05 50.001 50.05 50.01
NS 50.001 NS 50.05
50.05 50.05 NS NS
Shoot Root DCB Whole root
NS NS 50.001 50.001
NS NS 50.05 50.05
NS NS NS NS
Fe
4
P
2
The eect of pH and plaque on seedlings exposed to 0.5 mg l ÿ1 Cu
0
The presence of plaque had no eect on the Cu concentration of the DCB extract. However, there was signi®cantly more Cu in the extract at pH 6.0 than at pH 3.5 when iron plaque was absent (Fig. 3C, D). There were signi®cant eects of pH, plaque and the interaction between these treatments on Cu concentrations in roots (Table 1). Higher values were found when plaque was absent and at pH 6.0 (Fig. 3D). This was also true for shoot content but was not signi®cant for the eect of plaque alone. Greater concentrations of copper were also found in whole roots at pH 6.0 when plaque was absent.
K Si Al Ms Na
0
Cl
Ca
2
Ti
4
Mn
Cu
6
8
10
Range (keV)
2
Counts (×103)
A number of authors have attempted to characterize iron plaques that form on the roots of wetland plants in terms of both the chemistry/mineralogy and structure (Bacha and Hossner, 1977; Chen et al., 1980a,b; Taylor et al., 1984; Otte et al., 1989; St Cyr et al., 1993; Snowden and Wheeler, 1995). The use of diering techniques, plant species and laboratory and ®eld material has led to con¯icting information. This study has provided a unique comparison of laboratory- and ®eld-formed plaques on P. australis. The visual appearance of iron plaques in both environments was that of an amorphous granular deposit which incompletely covered the root surface. In previous studies it has been reported that plaque is distributed evenly across the root surface with distinct patterns of zonation (Chen et al., 1980b, Taylor et al., 1984; Otte et al., 1989; Snowden and Wheeler, 1995). This was also found in the present study with the majority of the plaque forming in the region 1 cm from the root tip. However, we have also demonstrated that plaque distribution may vary within this region with isolated clean areas found within these heavily plaqued zones. In both ®eld and laboratory samples there was no evidence of the formation of polyhedra or casts as reported previously (Chen et al., 1980b, Taylor et al., 1984), nor were deposits of iron found within the cells of the root. Although plaques formed under laboratory conditions were similar in structure and distribution, they diered in their chemistry. Under laboratory conditions the presence of iron and phos-
6
S
(n 3). NS, Non-signi®cant.
DISCUSSION
A
8
Counts (×102)
Cu
pH
10
B
Si
1
Al Fe Cl S Ms Na
0
0
P
Ca Mn
K Ti
2
4
Cu
6
8
10
Range (keV)
F I G . 2. Composition of plaques formed in laboratory conditions (A) ( pH 3.5) and ®eld conditions (B).
phorus indicated that the plaque was composed either of iron phosphate or of iron oxide with phosphorus adsorbed onto the surface. Iron is known to form iron phosphate complexes in other situations (Kuo, 1986) and these have already been documented in plaques (Snowden and Wheeler, 1995); however phosphorus was only present in the culture solution following plaque formation and is therefore more likely to have been adsorbed onto the plaque surface. In the ®eld specimens, phosphorus was not present in the plaque due to the extremely low concentrations of phosphorus in coal mine drainage waters. In these plaques the iron was associated with high levels of
Batty et al.ÐMetal Uptake in Phragmites australis
1500 1250 1000 750 500 250 0
2000
Cu concentration (mg l−1)
2000
A
Mn concentration (mg l−1)
1750
1750
Shoot
DCB
1250 1000 750 500 250 Shoot
DCB
Root Whole Root
B
1500 1250 1000 750 500 250
2000
C
1500
0
1750
0
Root Whole Root
Cu concentration (mg l−1)
Mn concentration (mg l−1)
2000
651
1750
Shoot
DCB
Root Whole Root
Shoot
DCB
Root Whole Root
D
1500 1250 1000 750 500 250 0
F I G . 3. Metal concentrations in P. australis seedlings exposed to 0.5 mg l ÿ1 Mn at pH 3.5 (A), 0.5 mg lÿ1 Mn at pH 6.0 (B), 0.5 mg l ÿ1 Cu at pH 3.5 (C), and 0.5 mg lÿ1 at pH 6.0 (D) in the presence (j) and absence (h) of plaque.
Al and Si. These two elements were probably present as clay particles and the iron was either adsorbed onto these particles or they were acting as a nucleus for iron oxide precipitation. A number of authors have suggested that the presence of iron plaque may act as a barrier to toxic metals (Otte et al., 1987, 1989; Greipsson and Crowder, 1992; Greipsson, 1994; Wang and Peverly, 1996). This may be achieved by adsorption onto iron compounds or co-precipitation. However, the majority of investigations have shown that the presence of plaque does not impede uptake (Benckiser et al., 1984; Levan and Riha, 1986; St Cyr and Crowder, 1987; Crowder and Coltman, 1993; Ye et al., 1997a, 1998), which contrasts with results from the present study. The data from the whole roots and shoots demonstrate that the presence of plaque reduces the uptake of Mn and Cu when plants are grown under higher pH conditions, but does not prevent it. The shoot concentrations of both Mn and Cu were elevated, reaching 371 and 357 mg kg ÿ1 d. wt, respectively. These values are much higher than previously reported in similar studies for Cu (St Cyr and Crowder, 1990; Ye et al., 1997a), but are lower than cited for Mn (Crowder and Coltman, 1993). The pH of the environment was also an important factor in controlling uptake of metals. The uptake of Mn in the shoots was lower at pH 3.5 than at 6.0 when plaque was present and for the whole roots in the presence and absence of plaque. At the lower pH there was a greater concentration of H ions around the root and these can eectively compete with other metal ions including Mn2 . This results in a strong inhibitory eect on the uptake of Mn
(Marschner, 1995). A similar result was found for Cu, although in the presence of plaque there was no signi®cant dierence between the pH conditions for all plant sections. In low pH conditions the presence of plaque did not have a signi®cant eect on the uptake of metals. Interference by H ions at low pH masks any potential eects of the presence of iron plaque. The mechanism by which plaque reduces the uptake of potentially phytotoxic metals may involve the adsorption of metals onto the plaque surface. From SEM analysis of both ®eld and laboratory specimens, it was evident that iron plaque may have an extremely limited capacity for the adsorption of other metals such as Mn or Cu. Neither of these metals was found in association with the iron plaque. In contrast to the EDS results, the analytical data show that Mn and Cu were present on the surface of the root, particularly at the higher pH (6.0), both with and without plaque. It has been suggested previously (Bacha and Hossner, 1977; McLaughlin et al., 1985) that the DCB extraction technique is harsh and may remove metals from within the root. In the absence of an iron plaque coating, this `stripping' could be accentuated due to the lack of a layer of protection. The results from the DCB extraction could therefore be an overestimation of the concentrations of metals; however, the detection limits for EDS analysis are lower than those for AAS, and therefore the metals may be present in the plaque but not detected by EDS. If the adsorption mechanism was active, then Cu and Mn should be found in higher concentrations in the plaque DCB extracts when levels were lower in the equivalent roots and shoots. However, this was not the case, and greater
652
Batty et al.ÐMetal Uptake in Phragmites australis
values of both Cu and Mn were present in the DCB extract when plaque was absent. Therefore, although the formation of iron plaque may inhibit metal uptake, a dierent mechanism must be operative, although this has not yet been identi®ed. These results contrast with many previous ®ndings which report the adsorption of metals onto the surface of plaques (St Cyr and Crowder, 1987; Crowder and St Cyr, 1991; Greipsson and Crowder, 1992; Greipsson, 1994; St Cyr and Campbell, 1996; Doyle and Otte, 1997; Ye et al., 1997a, 1998; Sundby et al., 1998). However, the majority of these studies have utilized the DCB extraction technique which may overestimate the concentrations of metals present and have not included data from other analytical sources. In addition, Mn has been reported to form Mn plaques (Bacha and Hossner, 1977; Crowder and Coltman, 1993), but in the present study there was no evidence of Mn plaque formation on the root surfaces. However, Mn plaque was found to form only when Mn was supplied at a concentration 41.0 mg l ÿ1 (Crowder and Coltman, 1993) and therefore it was unlikely to form under the conditions used here in which Mn was supplied at a concentration of 0.5 mg l ÿ1. At greater concentrations, Mn plaque may form and may aect the activity of other metals around the root of P. australis. Further investigations into the chemical composition of plaques under diering concentrations of metals need to be completed and these should include information from a variety of sources including chemical extraction, SEM and other microscopic techniques. CO N C L U S I O N S Structurally, iron plaques on the roots of ®eld- and laboratory-grown wetland plants appeared comparable. However, the chemistry of the resulting plaque is dependent upon the growth medium and it is therefore essential to accurately reproduce ®eld conditions in the laboratory. The use of chemical extraction techniques to investigate the chemistry of plaque deposits has been extensive in the past, but it is suggested that this may lead to an overestimation of metal concentrations. Results need to be interpreted in combination with additional data from EDS analysis and from whole root concentrations. Further research is also required to develop alternative extraction methods. When grown in higher pH conditions (6.0), iron plaques on the roots of P. australis reduce the uptake of Cu and Mn, but do not prevent it. The absence of Cu and Mn in the plaque deposits suggests that adsorption and co-precipitation are not important mechanisms in the prevention of uptake of these metals. Under lower pH (3.5) the activity of H ions interferes with the uptake of Cu and Mn and masks any potential eects of iron plaque. L I T E R AT U R E C I T E D Bacha RE, Hossner LR. 1977. Characteristics of coatings formed on rice roots as aected by iron and manganese additions. Soil Science Society of America Journal 41: 931±935.
Beining BA, Otte ML. 1996. Retention of metals originating from an abandoned lead±zinc mine by a wetland at Glendalough, Co. Wicklow. Proceedings of the Royal Irish Academy, Biology & Environment 96B: 117±126. Benckiser G, Santiago S, Neue HU, Watanabe I, Ottow JCG. 1984. Eect of fertilisation on exudation, dehydrogenase activity, ironreducing populations and Fe formation in the rhizosphere of rice (Oryza sativa L.) in relation to iron toxicity. Plant and Soil 79: 305±316. Chen CC, Dixon JB, Turner FT. 1980a. Iron coatings on roots: mineralogy and quantity in¯uencing factors. Soil Science Society of America Journal 44: 635±639. Chen CC, Dixon JB, Turner FT. 1980b. Iron coatings on rice roots: morphology and models of development. Soil Science Society of America Journal 44: 1113±1119. Crowder AA, Coltman DW. 1993. Formation of manganese oxide plaque on rice roots in solution culture under varying pH and manganese (Mn2 ) concentration conditions. Journal of Plant Nutrition 16: 589±599. Crowder A, St-Cyr L. 1991. Iron oxide plaques on wetland roots. Trends in Soil Science 1: 315±329. Crowder AA, Mac®e SM, Conlin T, St-Cyr L, Greipsson S. 1987. Iron hydroxide plaques on roots of wetland plants. In: Lindberg SE, Hutchinson TC, eds. Proceedings of the International Conference Heavy Metals in the Environment. Edinburgh: CEP Consultants, 404±406. Dodds-Smith ME, Payne CA, Gusek JJ. 1995. Reedbeds at Wheal Jane. Mining Environmental Management 22±24. Doyle MO, Otte ML. 1997. Organism-induced accumulation of iron, zinc and arsenic in wetland soils. Environmental Pollution 96: 1±11. Eger P, Lapakko K. 1989. Use of wetlands to remove nickel and copper from mine drainage. In: Hammer DA, ed. Constructed wetlands for wastewater treatment: municipal, industrial and agricultural. Michigan, USA: Lewis Publisher Inc, 780±787. Greipsson S. 1994. Eects of iron plaque on roots of rice on growth and metal concentration of seeds and plant tissues when cultivated in excess copper. Communications in Soil Science and Plant Analysis 25: 2761±2769. Greipsson S, Crowder AA. 1992. Amelioration of copper and nickel toxicity by iron plaque on roots of rice (Oryza sativa). Canadian Journal of Botany 70: 824±830. Hewitt EJ. 1966. Sand and water culture methods used in the study of plant nutrition. Technical Communication No 22, Commonwealth Agricultural Bureaux, Farnham Royal, Bucks. Jackson ML. 1958. Soil chemical analysisÐadvanced course. Madison, Wis: University of Wisconsin, 44±51. Kuo S. 1986. Concurrent sorption of phosphate and zinc, cadmium, or calcium by a hydrous ferric oxide. Soil Science Society of America Journal 50: 1412±1419. Lan C, Chen G, Li L, Wong MH. 1992. Use of cattails in treating wastewater from a Pb/Zn mine. Environmental Management 16: 75±80. Levan MA, Riha SJ. 1986. The precipitation of black oxide coatings on ¯ooded conifer roots of low internal porosity. Plant and Soil 95: 33±42. McLaughlin BE, Van Loon GW, Crowder AA. 1985. Comparison of selected washing treatments on Agrostis gigantea samples from mine tailings near Copper Cli, Ontario before analysis for Cu, Ni, Fe and K content. Plant and Soil 85: 433±465. Mac®e SM, Crowder AA. 1987. Soil factors in¯uencing ferric hydroxide plaque formation on roots of Typha latifolia L. Plant and Soil 102: 177±184. Marschner H. 1995. Mineral nutrition of higher plants. 2nd edn. London: Academic Press Ltd. Otte ML, Kearns CC, Doyle MO. 1995. Accumulation of arsenic and zinc in the rhizosphere of wetland plants. Bulletin of Environmental Contamination and Toxicology 55: 154±161. Otte ML, Buijs EP, Riemer L, Rozema J, Broekman RA. 1987. The iron-plaque on the roots of saltmarsh plants: a barrier to heavy metal uptake?. In: Lindberg SE, Hutchinson TC, eds. Proceedings of the International Conference Heavy Metals in the Environment. Edinburgh: CEP Consultants, 407±409.
Batty et al.ÐMetal Uptake in Phragmites australis Otte ML, Rozema J, Koster L, Haarsma MS, Broekman RA. 1989. Iron plaque on roots of Aster tripolium L.: interaction with zinc uptake. New Phytologist 111: 309±317. Scholes L, Shutes RBE, Revitt DM, Forshaw M, Purchase D. 1998. The treatment of metals in urban runo by constructed wetlands. The Science of the Total Environment 214: 211±219. Snowden RED, Wheeler BD. 1995. Chemical changes in selected wetland plant species with increasing Fe supply, with speci®c reference to root precipitates and Fe tolerance. New Phytologist 131: 503±520. St-Cyr L, Campbell PGC. 1996. Metals (Fe, Mn, Zn) in the root plaque of submerged aquatic plants collected in situ: relations with metal concentrations in the adjacent sediments and in the root tissue. Biogeochemistry 33: 45±76. St-Cyr L, Crowder AA. 1987. Relation between Fe, Mn, Cu and Zn in root plaque and leaves of Phragmites australis. In: Lindberg SE, Hutchinson TC, eds. International Conference on Heavy Metals in the Environment. Edinburgh: CEP Consultants, 466±468. St-Cyr L, Crowder AA. 1989. Factors aecting iron plaque on the roots of Phragmites australis (Cav.) Trin. ex Steudel. Plant and Soil 116: 85±93. St-Cyr L, Crowder AA. 1990. Manganese and copper in the root plaque of Phragmites australis (Cav.) Trin. ex Steudel. Soil Science 149: 191±198. St-Cyr L, Fortin D, Campbell PGC. 1993. Microscopic observations of the iron plaque of a submerged aquatic plant (Vallisneria americana Michx). Aquatic Botany 46: 155±167. Sundby B, Vale C, Cacador I, Catarino F, Madureira M-J, Caetano M. 1998. Metal-rich concretions on the roots of salt marsh plants: mechanisms and rate of formation. Limnology and Oceanography 43: 245±252.
653
Taylor GJ, Crowder AA. 1983. Use of the DCB technique for extraction of hydrous iron oxides from roots of wetland plants. American Journal of Botany 70: 1254±1257. Taylor GJ, Crowder AA, Rodden R. 1984. Formation and morphology of an iron plaque on the roots of Typha latifolia L. grown in solution culture. American Journal of Botany 71: 666±675. Wang T, Peverly JH. 1996. Oxidation states and fractionation of plaque iron on roots of common reeds. Soil Science Society of America Journal 60: 323±329. Wang T, Peverly JH. 1999. Iron oxidation states on root surfaces of a wetland plant (Phragmites australis). Soil Science Society of America Journal 63: 247±252. Wildeman TR, Laudon LS. 1989. Use of wetlands for treatment of environmental problems in mining: non-coal mining applications. In: Hammer DA, ed. Constructed wetlands for wastewater treatment; municipal, industrial and agricultural. Michigan, USA: Lewis Publ. Inc, 221±231. Ye ZH. 1995. Heavy metal tolerance, uptake and accumulation in populations of Typha latifolia L. and Phragmites australis (Cav.) Trin. ex Steudel. PhD Thesis, University of Sheeld, UK. Ye ZH, Baker AJM, Wong MH, Willis AJ. 1997a. Copper and nickel uptake, accumulation and tolerance in Typha latifolia with and without iron plaque on the root surface. New Phytologist 136: 481±488. Ye ZH, Baker AJM, Wong MH, Willis AJ. 1997b. Zinc, lead and cadmium tolerance, uptake and accumulation by Typha latifolia. New Phytologist 136: 469±480. Ye Z, Baker AJM, Wong MH, Willis AJ. 1998. Zinc, lead and cadmium accumulation and tolerance in Typha latifolia as aected by iron plaque on the root surface. Aquatic Botany 61: 55±67.
The Eect of pH and Plaque on the Uptake of Cu and Mn in Phragmites australis (Cav.) Trin ex. Steudel L . C . B AT T Y * {, A . J. M . B A K E R {, B . D. W H E E L E R{ and C . D . C U R T I S{ {Department of Plant and Animal Sciences, University of Sheeld, Sheeld S10 2TN, UK and {Department of Earth Sciences, University of Manchester, Manchester M13 9PL, UK Received: 10 August 1999 Returned for revision: 3 January 2000
Accepted: 24 March 2000
The physico-chemical properties of iron oxyhydroxide plaques formed on the roots of Phragmites australis under ®eld and laboratory conditions were determined using electron microscopy and energy dispersive spectrometry (EDS) analysis. Plaques were present as an amorphous coating on roots with an uneven distribution. They were shown to be composed of iron in both the ®eld and laboratory, and phosphorus was found to be adsorbed onto the surface of plaques formed in the laboratory. The uptake of copper and manganese in the presence and absence of plaque was investigated under two dierent pH regimes. Concentrations of Cu were lower in the shoots of P. australis in the presence of plaque (565 mg kg ÿ1) than when it was absent (1400 mg kg ÿ1), under growth conditions of higher pH (6.0). The adsorption of Cu and Mn onto the plaque surface was not the mechanism by which plaque reduced the uptake of other metals. Alternatively, the plaque may simply act as a physical barrier. Under conditions of lower pH (3.5) the activity of hydrogen ions at the root surface interfered with the movement of metals into the root and # 2000 Annals of Botany Company masked any potential eect of iron plaque. Key words: Phragmites australis, iron oxyhydroxide plaque, pH, manganese, copper, EDS, SEM, wetlands.
I N T RO D U C T I O N Acid mine drainage is a major source of metal contamination in many regions of the world. Discharges are characterized by low pH (53.5), elevated levels of metals such as Cu, Mn, Fe and Zn, together with low levels of organic compounds. For a number of years, wetlands, both natural and constructed, have been used to remove potentially toxic metals from contaminated drainage (Eger and Lapakko, 1989; Wildeman and Laudon, 1989; Lan et al., 1992; Dodds-Smith et al., 1995; Beining and Otte, 1996; Scholes et al., 1998). Wetland plant species, including Phragmites australis and Typha latifolia, are an important constituent of these passive treatments, but the strategy by which these species survive in highly metalliferous sites remains unclear. Coatings of iron (oxyhydr-)oxides known as `iron plaques' have been observed on a number of wetland plant species, including those growing in contaminated areas (Greipsson, 1994; Snowden and Wheeler, 1995; Sundby et al., 1998; Ye et al., 1998; Wang and Peverly, 1999). The exact cause of iron precipitation around roots is unknown but the oxidizing activity of roots, together with the action of micro-organisms, is thought to be involved. Iron oxide deposits are generally seen as an irregular porous coating on the root surface (Bacha and Hossner, 1977; St Cyr et al., 1993). Plaque deposits have been analysed using a variety of techniques including X-ray diraction and energy dispersive X-ray microanalysis. The coatings have been reported to consist of lepidocrocite * For correspondence. Fax 0191 222 6669, e-mail [email protected]
0305-7364/00/090647+07 $35.00/00
(g-FeOOH; Bacha and Hossner, 1977), a mixture of lepidocrocite and geothite (a-FeOOH; Chen et al., 1980a; St Cyr et al., 1993) and ferric phosphate (Snowden and Wheeler, 1995). Root plaques may also contain a variety of metals and metalloids which include aluminium, arsenic, cadmium, chromium, mercury, nickel, lead and zinc (Otte et al., 1987, 1989, 1995; St Cyr and Crowder, 1987, 1990; Greipsson and Crowder, 1992; St Cyr and Campbell, 1996; Doyle and Otte, 1997; Ye et al., 1997a, 1998; Sundby et al., 1998). The formation of iron plaques around the roots of wetland plants may constitute an adaptation to stressed environments. In ¯ooded areas the chemical reduction of iron and manganese compounds results in the accumulation of high levels of these bioavailable elements. In addition, there may be potentially phytotoxic levels of metals or metalloids, particularly in acid soils. Iron oxides present in soils and sediments have high speci®c surface areas and possess -OH functional groups which are capable of reacting with metals and other cations and anions (Kuo, 1986). It is possible that iron hydroxides forming on the roots of wetland plants have similar properties and may therefore immobilize and prevent the uptake of phytotoxic metals. This `exclusion' hypothesis is supported by reports of the amelioration of the toxic eects of phytotoxic metals in a variety of species. It has been reported that the presence of iron plaque on Oryza sativa seedlings improved growth under mildly toxic conditions of copper and (or) nickel exposure (Greipsson and Crowder, 1992; Greipsson, 1994). However, iron plaque also appeared to enhance the uptake of iron and it was proposed that the high concentration of # 2000 Annals of Botany Company
648
Batty et al.ÐMetal Uptake in Phragmites australis
iron in the leaves enabled competition by iron with copper for sensitive metabolic sites (Greipsson, 1994). Crowder et al. (1987) also found that the growth of Oryza sativa exposed to 0.5 ppm copper was greater in the presence of plaque. In contrast, investigations into the growth of Typha latifolia demonstrated that the presence of plaque on the roots of seedlings did not enhance metal tolerance or growth when subjected to copper and (or) nickel, zinc and (or) lead and cadmium (Ye, 1995; Ye et al., 1997b, 1998). However, the presence of plaque did reduce root length. Extensive research has also been undertaken to determine whether the presence of plaque reduces the uptake of phytotoxic metals. Iron plaques have been shown to act as a ®lter for the movement of iron, copper, zinc, nickel and cadmium into rhizomes and shoots (Greipsson and Crowder, 1992; Greipsson, 1994; Wang and Peverly, 1996). Otte et al. (1987, 1989) also found that a heavy coating of iron plaque may act as a barrier to zinc uptake in Aster tripolium L., but when the coating was light, zinc uptake was enhanced. The majority of research has shown that the formation of plaque does not impede the uptake of toxic metals. This has been demonstrated for the uptake of iron in Oryza sativa (Benckiser et al., 1984), manganese in Picea mariana Mill, Pinus resinosa Ait. (Levan and Riha, 1986) and Oryza sativa (Crowder and Coltman, 1993), copper and zinc in Phragmites australis (St Cyr and Crowder, 1987), copper and nickel in Typha latifolia (Ye et al., 1997a) and zinc and lead in Typha latifolia (Ye et al., 1998). Information in the literature is con¯icting, and the precise role of plaque remains unresolved. The research that has been undertaken has included investigation of seedlings from a wide variety of species including freshwater, saltwater and non-wetland plants, together with the use of material from both the ®eld and the laboratory. In the controlled conditions of the laboratory a number of techniques have been used with diering growth media (including nutrient solutions, sand and agar), a variety of pH conditions, diering metal sources (which has already been proved to be an important factor; Taylor et al., 1984), together with a number of dierent analytical techniques. It is therefore dicult to compare dierent investigations and to extract any de®nitive conclusions from the literature. The aim of this study was to determine the chemical and structural characteristics of iron plaques formed both in the ®eld and laboratory in order to assess the application of controlled laboratory experiments to the ®eld situation. In addition, an investigation was undertaken into the uptake of metals by the wetland species Phragmites australis in the presence and absence of plaque, in both acidic and more neutral pH conditions. The majority of investigations into the eects of plaque have been conducted in a narrow pH range between 5 and 7 (Mac®e and Crowder, 1987; St Cyr and Crowder, 1989); however wetland plants are frequently used to remediate acid mine drainage and therefore experiments need to be constructed in both acidic and neutral environments. A number of analytical techniques were used in order to achieve a more comprehensive set of results that could be compared.
M AT E R I A L S A ND M E T H O D S Seeds of Phragmites australis Trin. ex Steudel were collected from a non-contaminated site at Felixstowe, UK. The seeds were germinated at 218C and seedlings were grown supported on alkathene beads1 for 14 d in 10 % Rorison's solution (Hewitt, 1966). Seedlings of a uniform size were selected and ten were transplanted into blackened Perspex vessels (28 17 9 cm) containing 1.5 l of 10 % Rorison's solution ( pH 6.0). In total there were 36 units allowing for three replicates of all treatments, arranged in a randomized block design. The seedlings were grown for a further 63 d, the Rorison's solution being changed every 3 d. At the end of this period the pH of the solution in 18 of the containers was reduced gradually to 3.5 using 0.1 M H2SO4 . Each set of units at pH 3.5 and 6.0 were split into two subsets consisting of nine containers. In one of these subsets plaque formation was induced and in the remaining sets the roots were left clean. Plaque formation was induced on the roots of seedlings prior to metal treatment through the addition of 50 mg l ÿ1 of Fe ((NH4)2Fe(SO4)2 .6H2O) for 7 d in nutrient solution which did not contain potassium phosphate thereby preventing the interaction of Fe with P. In addition, the seedlings were placed in distilled water of the appropriate pH for 12 h before plaquing to prevent a similar interference of P. Plaque was visible on roots as an orange±brown deposit after 7 d. All seedlings were grown for a further 5 d on 10 % Rorison's solution prior to metal treatment. The two metals added were Cu (CuSO4 .5H2O) and Mn (MnSO4 .4H2O), both at a concentration of 0.5 mg l ÿ1. These were added to the appropriate containers to give the following conditions: (1) no plaque, Cu, pH 3.5; (2) no plaque, Cu, pH 6.0; (3) plaque, Cu, pH 3.5; (4) plaque, Cu, pH 6.0; (5) no plaque, Mn, pH 3.5; (6) no plaque, Mn, pH 6.0; (7) plaque, Mn, pH 3.5; and (8) plaque, Mn, pH 6.0. Control treatments contained no additional metals. During metal exposure, solutions were changed every 3 d. Roots were re-plaqued at 21 d and 50 d from the start of metal treatment to ensure that coverage of roots with plaque was maintained. Prior to the repeated exposure of roots to iron, the seedlings were placed in distilled water for 6 h before exposure to 50 mg l ÿ1 Fe for 3 d. At the end of the experiment (130 d) all plants were harvested, divided into roots and shoots and rinsed thoroughly in distilled water to remove dust contaminating the surface. A small subsample of root from each treatment was removed, freezedried and coated with carbon for scanning electron microscopy (SEM) analysis. The iron coating on the roots was removed using the dithionite±citrate±carbonate (DCB) method of Jackson (1958) as modi®ed by Taylor and Crowder (1983). Following DCB extraction, roots and shoots were dried at 408C and acid digested in 5 ml of 30 % HNO3 at 908C for a minimum of 8 h. The concentrations of Fe, Cu and Mn in the digests were determined by atomic absorption spectrophotometry (AAS). Results are presented for metal concentration in aerial parts (shoots), iron plaque (DCB extract), roots after plaque is removed (roots) and roots with plaque (whole roots). Whole root
Batty et al.ÐMetal Uptake in Phragmites australis
649
data were calculated from the root and DCB extract concentrations allowing for dierences in mass. Samples of P. australis were collected from a constructed reed bed receiving contaminated drainage from a derelict coal mine at Woolley Colliery, near Barnsley, UK. Root sections from these samples were prepared as outlined previously. Data on metal concentrations were analysed using a combination of t-tests and 2-way ANOVA followed by a Tukey test. Where necessary, data transformations were carried out, either loge or log10 . R E S U LT S Throughout the experiment all plants showed similar growth and development of both roots and shoots. Laboratory plaques Morphology. The plaque deposits were clearly visible as orange±brown coatings on the root surface, extending from 1 cm behind the growing tip along the entire length of the root. Under the SEM these deposits could be distinguished by textural dierences from the unplaqued root. Figure 1A shows a section of the plaqued root surface when grown without exposure to Mn or Cu. It is evident that plaque was present as a particulate deposit that follows the contours of cells. The coating was not uniform and plaque deposits were absent from large areas of the root surface. Chen et al. (1980b) reported the presence of cell casts and polyhedra in plaque deposits, but these were not found on roots in the present study. No evidence of penetration of the plaque into the epidermal cells was found when transverse sections of the root were studied. In the absence of iron and the presence of Mn there was no formation of manganese plaques. Chemical composition. In clean areas of the root, X-ray analysis revealed the presence of K, S and lesser amounts of Ca, P and Cl which is a residual signature of the nutrient solution. The precipitates formed in the presence of Fe had a very speci®c chemical signature and were composed of Fe and P, together with lesser amounts of S and K (Fig. 2A). It is likely that the plaques were composed of iron phosphate. In the presence of Cu or Mn the plaques had the same chemical signature, but there was no evidence of either Cu or Mn within the deposits. Field plaques Morphology. In equivalent root sections of P. australis collected from the ®eld, plaque was also present as an orange±brown deposit on the root surface, extending from 1 cm behind the root tip along the entire length of the root. Under SEM it was evident that these particulate deposits had a similar form to those found on laboratory samples (Fig. 1B). The plaque deposits were present as amorphous particulate coverings of the root surface but were not ubiquitous, and plaque was not evident in some areas. Cell
F I G . 1. Micrographs of P. australis roots grown in a controlled laboratory (A) (500), and ®eld conditions (B) (600). IP, Iron plaque; C, clean, unplaqued areas.
casts and polyhedra were not present and there was no evidence of penetration into epidermal cells. Chemical composition. The chemistry of the plaque deposits from the ®eld diered from that of the laboratory samples. The iron precipitates present were composed mainly of iron and silica with accessory elements including Al and Mn (Fig. 2B). The absence of phosphorus implied that the plaques were not composed of iron phosphates but that iron was forming a dierent compound, yet to be identi®ed. In some regions of the root surface, the plaques had a similar chemistry, but Mn was also present in greater quantities than iron. Copper was present, associated with the iron plaque in isolated areas of the root. The eect of pH and plaque on seedlings exposed to 0.5 mg l ÿ1 Mn No signi®cant eects of pH or plaque were found for root or shoot contents (Table 1). However, at higher pH a signi®cantly greater concentration of Mn was found in the DCB extract when plaque was absent than when it was present (Fig. 3A). This was also true for the whole root data. When plaque was absent there was also more Mn in the DCB and whole root extracts at pH 6.0 (Fig. 3B).
650
Batty et al.ÐMetal Uptake in Phragmites australis
T A B L E 1. Signi®cance levels of pH and plaque treatments according to two-way analysis of variance for dierent plant fractions shown in Fig. 3
Mn
Plaque
Interaction
Shoot Root DCB Whole root
50.05 50.001 50.05 50.01
NS 50.001 NS 50.05
50.05 50.05 NS NS
Shoot Root DCB Whole root
NS NS 50.001 50.001
NS NS 50.05 50.05
NS NS NS NS
Fe
4
P
2
The eect of pH and plaque on seedlings exposed to 0.5 mg l ÿ1 Cu
0
The presence of plaque had no eect on the Cu concentration of the DCB extract. However, there was signi®cantly more Cu in the extract at pH 6.0 than at pH 3.5 when iron plaque was absent (Fig. 3C, D). There were signi®cant eects of pH, plaque and the interaction between these treatments on Cu concentrations in roots (Table 1). Higher values were found when plaque was absent and at pH 6.0 (Fig. 3D). This was also true for shoot content but was not signi®cant for the eect of plaque alone. Greater concentrations of copper were also found in whole roots at pH 6.0 when plaque was absent.
K Si Al Ms Na
0
Cl
Ca
2
Ti
4
Mn
Cu
6
8
10
Range (keV)
2
Counts (×103)
A number of authors have attempted to characterize iron plaques that form on the roots of wetland plants in terms of both the chemistry/mineralogy and structure (Bacha and Hossner, 1977; Chen et al., 1980a,b; Taylor et al., 1984; Otte et al., 1989; St Cyr et al., 1993; Snowden and Wheeler, 1995). The use of diering techniques, plant species and laboratory and ®eld material has led to con¯icting information. This study has provided a unique comparison of laboratory- and ®eld-formed plaques on P. australis. The visual appearance of iron plaques in both environments was that of an amorphous granular deposit which incompletely covered the root surface. In previous studies it has been reported that plaque is distributed evenly across the root surface with distinct patterns of zonation (Chen et al., 1980b, Taylor et al., 1984; Otte et al., 1989; Snowden and Wheeler, 1995). This was also found in the present study with the majority of the plaque forming in the region 1 cm from the root tip. However, we have also demonstrated that plaque distribution may vary within this region with isolated clean areas found within these heavily plaqued zones. In both ®eld and laboratory samples there was no evidence of the formation of polyhedra or casts as reported previously (Chen et al., 1980b, Taylor et al., 1984), nor were deposits of iron found within the cells of the root. Although plaques formed under laboratory conditions were similar in structure and distribution, they diered in their chemistry. Under laboratory conditions the presence of iron and phos-
6
S
(n 3). NS, Non-signi®cant.
DISCUSSION
A
8
Counts (×102)
Cu
pH
10
B
Si
1
Al Fe Cl S Ms Na
0
0
P
Ca Mn
K Ti
2
4
Cu
6
8
10
Range (keV)
F I G . 2. Composition of plaques formed in laboratory conditions (A) ( pH 3.5) and ®eld conditions (B).
phorus indicated that the plaque was composed either of iron phosphate or of iron oxide with phosphorus adsorbed onto the surface. Iron is known to form iron phosphate complexes in other situations (Kuo, 1986) and these have already been documented in plaques (Snowden and Wheeler, 1995); however phosphorus was only present in the culture solution following plaque formation and is therefore more likely to have been adsorbed onto the plaque surface. In the ®eld specimens, phosphorus was not present in the plaque due to the extremely low concentrations of phosphorus in coal mine drainage waters. In these plaques the iron was associated with high levels of
Batty et al.ÐMetal Uptake in Phragmites australis
1500 1250 1000 750 500 250 0
2000
Cu concentration (mg l−1)
2000
A
Mn concentration (mg l−1)
1750
1750
Shoot
DCB
1250 1000 750 500 250 Shoot
DCB
Root Whole Root
B
1500 1250 1000 750 500 250
2000
C
1500
0
1750
0
Root Whole Root
Cu concentration (mg l−1)
Mn concentration (mg l−1)
2000
651
1750
Shoot
DCB
Root Whole Root
Shoot
DCB
Root Whole Root
D
1500 1250 1000 750 500 250 0
F I G . 3. Metal concentrations in P. australis seedlings exposed to 0.5 mg l ÿ1 Mn at pH 3.5 (A), 0.5 mg lÿ1 Mn at pH 6.0 (B), 0.5 mg l ÿ1 Cu at pH 3.5 (C), and 0.5 mg lÿ1 at pH 6.0 (D) in the presence (j) and absence (h) of plaque.
Al and Si. These two elements were probably present as clay particles and the iron was either adsorbed onto these particles or they were acting as a nucleus for iron oxide precipitation. A number of authors have suggested that the presence of iron plaque may act as a barrier to toxic metals (Otte et al., 1987, 1989; Greipsson and Crowder, 1992; Greipsson, 1994; Wang and Peverly, 1996). This may be achieved by adsorption onto iron compounds or co-precipitation. However, the majority of investigations have shown that the presence of plaque does not impede uptake (Benckiser et al., 1984; Levan and Riha, 1986; St Cyr and Crowder, 1987; Crowder and Coltman, 1993; Ye et al., 1997a, 1998), which contrasts with results from the present study. The data from the whole roots and shoots demonstrate that the presence of plaque reduces the uptake of Mn and Cu when plants are grown under higher pH conditions, but does not prevent it. The shoot concentrations of both Mn and Cu were elevated, reaching 371 and 357 mg kg ÿ1 d. wt, respectively. These values are much higher than previously reported in similar studies for Cu (St Cyr and Crowder, 1990; Ye et al., 1997a), but are lower than cited for Mn (Crowder and Coltman, 1993). The pH of the environment was also an important factor in controlling uptake of metals. The uptake of Mn in the shoots was lower at pH 3.5 than at 6.0 when plaque was present and for the whole roots in the presence and absence of plaque. At the lower pH there was a greater concentration of H ions around the root and these can eectively compete with other metal ions including Mn2 . This results in a strong inhibitory eect on the uptake of Mn
(Marschner, 1995). A similar result was found for Cu, although in the presence of plaque there was no signi®cant dierence between the pH conditions for all plant sections. In low pH conditions the presence of plaque did not have a signi®cant eect on the uptake of metals. Interference by H ions at low pH masks any potential eects of the presence of iron plaque. The mechanism by which plaque reduces the uptake of potentially phytotoxic metals may involve the adsorption of metals onto the plaque surface. From SEM analysis of both ®eld and laboratory specimens, it was evident that iron plaque may have an extremely limited capacity for the adsorption of other metals such as Mn or Cu. Neither of these metals was found in association with the iron plaque. In contrast to the EDS results, the analytical data show that Mn and Cu were present on the surface of the root, particularly at the higher pH (6.0), both with and without plaque. It has been suggested previously (Bacha and Hossner, 1977; McLaughlin et al., 1985) that the DCB extraction technique is harsh and may remove metals from within the root. In the absence of an iron plaque coating, this `stripping' could be accentuated due to the lack of a layer of protection. The results from the DCB extraction could therefore be an overestimation of the concentrations of metals; however, the detection limits for EDS analysis are lower than those for AAS, and therefore the metals may be present in the plaque but not detected by EDS. If the adsorption mechanism was active, then Cu and Mn should be found in higher concentrations in the plaque DCB extracts when levels were lower in the equivalent roots and shoots. However, this was not the case, and greater
652
Batty et al.ÐMetal Uptake in Phragmites australis
values of both Cu and Mn were present in the DCB extract when plaque was absent. Therefore, although the formation of iron plaque may inhibit metal uptake, a dierent mechanism must be operative, although this has not yet been identi®ed. These results contrast with many previous ®ndings which report the adsorption of metals onto the surface of plaques (St Cyr and Crowder, 1987; Crowder and St Cyr, 1991; Greipsson and Crowder, 1992; Greipsson, 1994; St Cyr and Campbell, 1996; Doyle and Otte, 1997; Ye et al., 1997a, 1998; Sundby et al., 1998). However, the majority of these studies have utilized the DCB extraction technique which may overestimate the concentrations of metals present and have not included data from other analytical sources. In addition, Mn has been reported to form Mn plaques (Bacha and Hossner, 1977; Crowder and Coltman, 1993), but in the present study there was no evidence of Mn plaque formation on the root surfaces. However, Mn plaque was found to form only when Mn was supplied at a concentration 41.0 mg l ÿ1 (Crowder and Coltman, 1993) and therefore it was unlikely to form under the conditions used here in which Mn was supplied at a concentration of 0.5 mg l ÿ1. At greater concentrations, Mn plaque may form and may aect the activity of other metals around the root of P. australis. Further investigations into the chemical composition of plaques under diering concentrations of metals need to be completed and these should include information from a variety of sources including chemical extraction, SEM and other microscopic techniques. CO N C L U S I O N S Structurally, iron plaques on the roots of ®eld- and laboratory-grown wetland plants appeared comparable. However, the chemistry of the resulting plaque is dependent upon the growth medium and it is therefore essential to accurately reproduce ®eld conditions in the laboratory. The use of chemical extraction techniques to investigate the chemistry of plaque deposits has been extensive in the past, but it is suggested that this may lead to an overestimation of metal concentrations. Results need to be interpreted in combination with additional data from EDS analysis and from whole root concentrations. Further research is also required to develop alternative extraction methods. When grown in higher pH conditions (6.0), iron plaques on the roots of P. australis reduce the uptake of Cu and Mn, but do not prevent it. The absence of Cu and Mn in the plaque deposits suggests that adsorption and co-precipitation are not important mechanisms in the prevention of uptake of these metals. Under lower pH (3.5) the activity of H ions interferes with the uptake of Cu and Mn and masks any potential eects of iron plaque. L I T E R AT U R E C I T E D Bacha RE, Hossner LR. 1977. Characteristics of coatings formed on rice roots as aected by iron and manganese additions. Soil Science Society of America Journal 41: 931±935.
Beining BA, Otte ML. 1996. Retention of metals originating from an abandoned lead±zinc mine by a wetland at Glendalough, Co. Wicklow. Proceedings of the Royal Irish Academy, Biology & Environment 96B: 117±126. Benckiser G, Santiago S, Neue HU, Watanabe I, Ottow JCG. 1984. Eect of fertilisation on exudation, dehydrogenase activity, ironreducing populations and Fe formation in the rhizosphere of rice (Oryza sativa L.) in relation to iron toxicity. Plant and Soil 79: 305±316. Chen CC, Dixon JB, Turner FT. 1980a. Iron coatings on roots: mineralogy and quantity in¯uencing factors. Soil Science Society of America Journal 44: 635±639. Chen CC, Dixon JB, Turner FT. 1980b. Iron coatings on rice roots: morphology and models of development. Soil Science Society of America Journal 44: 1113±1119. Crowder AA, Coltman DW. 1993. Formation of manganese oxide plaque on rice roots in solution culture under varying pH and manganese (Mn2 ) concentration conditions. Journal of Plant Nutrition 16: 589±599. Crowder A, St-Cyr L. 1991. Iron oxide plaques on wetland roots. Trends in Soil Science 1: 315±329. Crowder AA, Mac®e SM, Conlin T, St-Cyr L, Greipsson S. 1987. Iron hydroxide plaques on roots of wetland plants. In: Lindberg SE, Hutchinson TC, eds. Proceedings of the International Conference Heavy Metals in the Environment. Edinburgh: CEP Consultants, 404±406. Dodds-Smith ME, Payne CA, Gusek JJ. 1995. Reedbeds at Wheal Jane. Mining Environmental Management 22±24. Doyle MO, Otte ML. 1997. Organism-induced accumulation of iron, zinc and arsenic in wetland soils. Environmental Pollution 96: 1±11. Eger P, Lapakko K. 1989. Use of wetlands to remove nickel and copper from mine drainage. In: Hammer DA, ed. Constructed wetlands for wastewater treatment: municipal, industrial and agricultural. Michigan, USA: Lewis Publisher Inc, 780±787. Greipsson S. 1994. Eects of iron plaque on roots of rice on growth and metal concentration of seeds and plant tissues when cultivated in excess copper. Communications in Soil Science and Plant Analysis 25: 2761±2769. Greipsson S, Crowder AA. 1992. Amelioration of copper and nickel toxicity by iron plaque on roots of rice (Oryza sativa). Canadian Journal of Botany 70: 824±830. Hewitt EJ. 1966. Sand and water culture methods used in the study of plant nutrition. Technical Communication No 22, Commonwealth Agricultural Bureaux, Farnham Royal, Bucks. Jackson ML. 1958. Soil chemical analysisÐadvanced course. Madison, Wis: University of Wisconsin, 44±51. Kuo S. 1986. Concurrent sorption of phosphate and zinc, cadmium, or calcium by a hydrous ferric oxide. Soil Science Society of America Journal 50: 1412±1419. Lan C, Chen G, Li L, Wong MH. 1992. Use of cattails in treating wastewater from a Pb/Zn mine. Environmental Management 16: 75±80. Levan MA, Riha SJ. 1986. The precipitation of black oxide coatings on ¯ooded conifer roots of low internal porosity. Plant and Soil 95: 33±42. McLaughlin BE, Van Loon GW, Crowder AA. 1985. Comparison of selected washing treatments on Agrostis gigantea samples from mine tailings near Copper Cli, Ontario before analysis for Cu, Ni, Fe and K content. Plant and Soil 85: 433±465. Mac®e SM, Crowder AA. 1987. Soil factors in¯uencing ferric hydroxide plaque formation on roots of Typha latifolia L. Plant and Soil 102: 177±184. Marschner H. 1995. Mineral nutrition of higher plants. 2nd edn. London: Academic Press Ltd. Otte ML, Kearns CC, Doyle MO. 1995. Accumulation of arsenic and zinc in the rhizosphere of wetland plants. Bulletin of Environmental Contamination and Toxicology 55: 154±161. Otte ML, Buijs EP, Riemer L, Rozema J, Broekman RA. 1987. The iron-plaque on the roots of saltmarsh plants: a barrier to heavy metal uptake?. In: Lindberg SE, Hutchinson TC, eds. Proceedings of the International Conference Heavy Metals in the Environment. Edinburgh: CEP Consultants, 407±409.
Batty et al.ÐMetal Uptake in Phragmites australis Otte ML, Rozema J, Koster L, Haarsma MS, Broekman RA. 1989. Iron plaque on roots of Aster tripolium L.: interaction with zinc uptake. New Phytologist 111: 309±317. Scholes L, Shutes RBE, Revitt DM, Forshaw M, Purchase D. 1998. The treatment of metals in urban runo by constructed wetlands. The Science of the Total Environment 214: 211±219. Snowden RED, Wheeler BD. 1995. Chemical changes in selected wetland plant species with increasing Fe supply, with speci®c reference to root precipitates and Fe tolerance. New Phytologist 131: 503±520. St-Cyr L, Campbell PGC. 1996. Metals (Fe, Mn, Zn) in the root plaque of submerged aquatic plants collected in situ: relations with metal concentrations in the adjacent sediments and in the root tissue. Biogeochemistry 33: 45±76. St-Cyr L, Crowder AA. 1987. Relation between Fe, Mn, Cu and Zn in root plaque and leaves of Phragmites australis. In: Lindberg SE, Hutchinson TC, eds. International Conference on Heavy Metals in the Environment. Edinburgh: CEP Consultants, 466±468. St-Cyr L, Crowder AA. 1989. Factors aecting iron plaque on the roots of Phragmites australis (Cav.) Trin. ex Steudel. Plant and Soil 116: 85±93. St-Cyr L, Crowder AA. 1990. Manganese and copper in the root plaque of Phragmites australis (Cav.) Trin. ex Steudel. Soil Science 149: 191±198. St-Cyr L, Fortin D, Campbell PGC. 1993. Microscopic observations of the iron plaque of a submerged aquatic plant (Vallisneria americana Michx). Aquatic Botany 46: 155±167. Sundby B, Vale C, Cacador I, Catarino F, Madureira M-J, Caetano M. 1998. Metal-rich concretions on the roots of salt marsh plants: mechanisms and rate of formation. Limnology and Oceanography 43: 245±252.
653
Taylor GJ, Crowder AA. 1983. Use of the DCB technique for extraction of hydrous iron oxides from roots of wetland plants. American Journal of Botany 70: 1254±1257. Taylor GJ, Crowder AA, Rodden R. 1984. Formation and morphology of an iron plaque on the roots of Typha latifolia L. grown in solution culture. American Journal of Botany 71: 666±675. Wang T, Peverly JH. 1996. Oxidation states and fractionation of plaque iron on roots of common reeds. Soil Science Society of America Journal 60: 323±329. Wang T, Peverly JH. 1999. Iron oxidation states on root surfaces of a wetland plant (Phragmites australis). Soil Science Society of America Journal 63: 247±252. Wildeman TR, Laudon LS. 1989. Use of wetlands for treatment of environmental problems in mining: non-coal mining applications. In: Hammer DA, ed. Constructed wetlands for wastewater treatment; municipal, industrial and agricultural. Michigan, USA: Lewis Publ. Inc, 221±231. Ye ZH. 1995. Heavy metal tolerance, uptake and accumulation in populations of Typha latifolia L. and Phragmites australis (Cav.) Trin. ex Steudel. PhD Thesis, University of Sheeld, UK. Ye ZH, Baker AJM, Wong MH, Willis AJ. 1997a. Copper and nickel uptake, accumulation and tolerance in Typha latifolia with and without iron plaque on the root surface. New Phytologist 136: 481±488. Ye ZH, Baker AJM, Wong MH, Willis AJ. 1997b. Zinc, lead and cadmium tolerance, uptake and accumulation by Typha latifolia. New Phytologist 136: 469±480. Ye Z, Baker AJM, Wong MH, Willis AJ. 1998. Zinc, lead and cadmium accumulation and tolerance in Typha latifolia as aected by iron plaque on the root surface. Aquatic Botany 61: 55±67.