Availability of Ferrocyanide and Ferricyanide ... - Springer Link

Arch Environ Contam Toxicol (2008) 55:229–237 DOI 10.1007/s00244-007-9101-6

Availability of Ferrocyanide and Ferricyanide Complexes as a Nitrogen Source to Cyanogenic Plants Xiao-Zhang Yu Æ Ji-Dong Gu Æ Tian-Peng Li

Received: 30 July 2007 / Accepted: 22 November 2007 / Published online: 8 January 2008 Ó Springer Science+Business Media, LLC 2008

Abstract The effects of additional nitrogen on the toxicity and removal of ferrocyanide and ferricyanide by cyanogenic plants were investigated. Maize (Zea mays L. var. ZN 304) seedlings were grown in the hydroponic solutions with or without additional nitrogen, and amended with either potassium ferrocyanide or potassium ferricyanide at 25.0 ± 0.5°C for 144 h. Various physiological parameters were monitored to determine the responses of the plant seedlings to the exposure of these two chemicals. A remarkable decrease in transpiration rate, biomass, shoot length, chlorophyll contents, and soluble proteins was evident for maize seedlings grown in the N-free hydroponic solutions spiked with either ferrocyanide or ferricyanide (P \ 0.01), but slight changes were observed in the selective parameters in the N-containing hydroponic solutions spiked with either of these chemicals (P [ 0.05). A higher removal of ferrocyanide than ferricyanide was registered in the N-free hydroponic solutions, but more ferricyanide than ferrocyanide was removed by maize grown in the N-containing nutrient solutions (P \ 0.01). Although roots of maize accumulated iron cyanides, more cyanide was recovered in plant materials of those grown in the N-containing hydroponic solutions than the N-free

X.-Z. Yu J.-D. Gu (&) Laboratory of Environmental Toxicology, School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, People’s Republic of China e-mail: [email protected] T.-P. Li Department of Environmental Science & Engineering, Hunan Agricultural University, Changsha 41028, People’s Republic of China

nutrient solutions (P \ 0.05). Mass balance analysis indicated that the majority of the iron cyanides removed from solution was assimilated by maize and additional nitrogen had a significantly negative impact on the uptake of both chemicals (P \ 0.01). Results of this study suggest that uptake and assimilation mechanisms for ferrocyanide and ferricyanide might be quite different in maize and the application of the external nitrogen has a substantial influence on the removal of both iron cyanides by plants. None of the iron cyanide complexes can serve as a sole nitrogen source to support maize growth.

The commercial use of cyanide as a leaching reagent for the extraction of metals from mineral ores can be tracked to New Zealand over one century ago. Cyanide is widely used in the extraction of gold and silver, and in the production of adhesives, computer electronics, fire retardants, cosmetics, dyes, nylon, paints, pharmaceuticals, plexiglass, rocket propellant, road and table salts, and marzipan (Mudder and Botz 2001). It has been estimated that the annual production of hydrogen cyanide (HCN) is 1.4 million metric tons and more than 100,000 tons of cyanide enters the environment annually (Mudder and Botz 2001). The occurrence of cyanide associated with gold mining has increased the ecological risk of soils, sediments, and surface and ground water (Korte et al. 2000). Cyanide in the environment frequently complexes with many metals, forming a variety of metal cyanide complexes, 3 III e.g., ferrocyanide FeII ðCNÞ4 6 , ferricyanide Fe ðCNÞ6 , III II ferroferricyanide (Prussian blue) Fe4 [Fe (CN)6]3 are the common and stable forms (Theis et al. 1994; Meeussen et al. 1992), which accounted for more than 97% of the total cyanide in soils and groundwater (Theis et al. 1994).

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The most conclusive information is that plants can metabolize free cyanide by the enzyme beta-cyanoalanine synthase (Blumenthal et al. 1968) and the final metabolite is asparagine, an important amino acid (Castirc et al. 1972). One possible fate of cyanide is the oxidation of CN- to CO2 via monooxygenase or dioxygenase systems (Ebel et al. 2006). Another possible fate is the photooxidation of cyanide to form cyanate (Nowakowska et al. 1999), which could be further oxidized to CO2 by cyanases. These enzymes have been identified in plants and microorganisms (Guilloton et al. 2002). Photodecomposition of iron cyanide complexes to free cyanide often occurs when present in the vadose zone or being discharged into surface waters (Kjeldsen 1999; Samiotakis and Ebbs 2004). Degradation by microorganisms has also been reported. The fungus Fusarium solani is able to use metal cyanide for growth and biotransforms ferrocyanide to ammonia under neutral and acid conditions (Barclay et al. 1998). Yeast cells of Saccharomyces cerevisiae reduce ferricyanide either through the ferrireductases involved in iron transfer systems (Lessuisse et al. 1995) or through the NADH-dependent menadione reductase (Yashiki and Yamashoji 1996). Phytoremediation of free cyanide by a number of plants from three continents and climate zones (Ebbs et al. 2003; Larsen et al. 2005; Yu et al. 2004) has exhibited much promise for the remediation of cyanide from contaminated sediment and groundwater. Although iron cyanide complexes have long been considered membrane impermeable (Roustan and Sablayrolles 2003), uptake of ferrocyanide and ferricyanide by plants has been investigated by Ebbs et al. (2003), Samiotakis and Ebbs (2004), Larsen and Trapp (2006), and Yu et al. (2006) and was probably followed by metabolism inside plants (Ebbs et al. 2003; Larsen and Trapp 2006). Federico and Giartosio (1983) confirmed that the existence of a NADH-ferricyanide (O2) electron transport system, located within the plasmalemma, links to active reduction of ferricyanide in maize (Zea mays L., var XL 342). In a recent work by Kang et al. (2007), approximately 19% of the iron cyanides (Prussian blue) in the cyanogenic plant species, sorghum (Sorghum bicolor var P 721) and flax (Linum usitassimum var Omega-Gold), was transformed, but 7% of the 14C-labeled cyanide was converted to 14CO2 by sorghum and 6% by flax and a small amount of unaltered cyanide was shown to be accumulated by the plants. Although plants can metabolize free cyanide to an amino acid and take up iron cyanide complexes, little convincing information is available about iron cyanide complexes as potential nitrogen source to support the growth of any plants. The objectives of this investigation were to ascertain the uptake, assimilation, and accumulation of ferrocyanide and ferricyanide in cyanogenic plant

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Arch Environ Contam Toxicol (2008) 55:229–237

species maize due to the application of the external nitrogen.

Materials and Methods Plant Specimens and Exposure Regimes Maize (Zea mays L. var. ZN 304) seeds from the Hunan Academy of Agricultural Sciences, P.R. China after cleaning were planted under laboratory condition with a constant temperature of 25°C until shoots appeared. After another 192 h of growth, the young maize seedlings were used in the subsequent experiments. Each maize seedling was transferred to a 50 mL Erlenmeyer flask filled with approximately 50 mL modified ISO 8692 standard nutrient solution (Table 1). The flasks were all wrapped with aluminum foil to prevent escape of water, and to inhibit potential growth of algae. For each treatment, nine replicates were prepared. All flasks were housed in a climate control chamber maintained at a constant temperature of 25.0 ± 0.5°C and a relative humidity of 60 ± 2% under continuous artificial light. The plants were conditioned for 48 h first to adapt to the environmental conditions. Then, the weight of the plant–flask system was measured and recorded individually. Each flask including the young maize seedlings was weighed again after 24 h to determine the transpiration rate. Plants with a similar transpiration rate were selected for the tests. The nutrient solution in each flask was then replaced by spiked solution. Potassium ferricyanide [K3Fe(CN)6] or potassium ferrocyanide [K4Fe(CN)6] of analytical grade with C 95% purity were used. It should be noted that 1 mg K3Fe(CN)6 and K4Fe(CN)6 equals 0.474 and 0.424 mg CN, respectively. The initial concentrations of spiked solutions were 10.40 (±0.536) and 11.74 (±0.653) mg CN/L for the treatments with ferrocyanide and ferricyanide, respectively. Six different treatments were prepared for each test chemical and their respective controls: (S1) 20% strength N-free nutrient solution; (S2) 40% strength N-free nutrient solution; (S3) Table 1 Chemical composition of the modified ISO 8692 nutrient solution used in this study Macronutrients (lmol/L) NaNO*3

Micronutrients (nmol/L)

2823.9

H3BO3

2992.1

MnCl24H2O

2097.0

MgCl26H2O

59.0

CaCl22H2O

122.4

MgSO47H2O

60.9

CoCl26H2O

6.3

KH2PO4

246.0

CuCl22H2O

0.1

NaHCO3

1785.5

*

Only used in S6

ZnCl2

NaMoO42H2O

22.0

28.9


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60% strength N-free nutrient solution; (S4) 80% strength N-free nutrient solution; (S5) 100% strength N-free nutrient solution; (S6) N-containing nutrient solution. Two controls of six replicates were made, both without test chemicals. The first control included maize grown in the Ncontaining nutrient solution and the other was in the 100% strength N-free nutrient solution to quantify the change of physiological parameters selected in this study. Additional controls with test chemicals, but without maize were made to track the abiotic reduction of iron cyanides in the hydroponic solution over the period of experiments.

Determination of the Transpiration Rate

Chlorophyll Measurement

The assimilation capacity of iron cyanide complexes vp (lg CN/g FW.d) was calculated from

The chlorophyll content in leaves was determined spectrophotometrically at the end of the experiments (144 h). Plant leaves were cut into small pieces, precisely weighed (0.5 g fresh weight) and placed in 25 mL flasks. Then, 80% acetone was added to the mark of 25 mL. Three separate flasks were conducted for each treatment group. All flasks were placed in the dark for 24 h. During this period, flasks were shaken twice intermittently. The absorption of light at 645 and 663 nm was measured in a curvette with an optical path of 10 mm against 80% acetone as a blank control. The amount of chlorophyll a and chlorophyll b in plant leaves was calculated by Maclachalam and Zalik’s equation (1963).

Soluble Protein Measurement The soluble protein content was determined spectrophotometrically in fresh leaves using the top shoot, as described by Jin and Ding (1981). At the end of the experiments (144 h), 0.5 g of the tissue materials (fresh weight) was precisely weighed and placed in a triturator. Before trituration, 2.5 mL of 65 mM phosphate buffer solution (pH 7.8) containing 0.4% mercapto-ethanol (v/v) was added. Trituration was performed in an ice bath followed by centrifuging at 12000 9 g for 15 min. The supernatant was collected and stored at 4°C for analysis of the soluble protein in leaves. At the time of the analysis, 0.1 mL aliquot of the supernatant samples was pippetted into a vessel and 5 mL Coomassie Brilliant Blue G-250 solution (Sigma-Aldrich Inc., St. Louis, Missouri) was added. After thorough mixing, the vessel was left standing for 2 min. The absorption of light at 595 nm was measured spectrophotometrically against water as reference. Albumin bovine V solution from bovine serum (Sigma-Aldrich Inc., St. Louis, Missouri) was used as a standard. Three separate flasks were conducted for each treatment group.

Inhibition of transpiration is a rapid measure for the toxic effect of a chemical or a substrate to trees (Trapp et al. 2000). The effect of iron cyanide complexes on maize seedlings was quantified by measuring the transpiration rate of maize in flasks. The weight loss of the plant–flask system was expressed as the transpiration rate.

Determination of the Assimilation Rates of Iron Cyanide Complexes

vp ¼

MðinitialÞ MðfinalÞ MðrootÞ MðshootÞ WðplantÞ DT

Where M(initial), M(final), M(root), and M(shoot) are the total cyanide (lg) in hydroponic solution and in different plant materials. W(plant) is the biomass of the plant (g), and DT is the time period (d).

Chemical Analysis Total Cyanide in Water Total cyanide is the sum of easily liberated cyanide (HCN and CN–) and complexed cyanide. The total cyanide in the solution was analyzed by a standard method (State Environmental Protection Administration of P.R. China, 1989, method number GB 7487–87). Ten milliters of 1% NaOH were added to the reservoir vessel of the distillation unit. Five milliliters of the spiked solution were placed in a 500 mL round-bottomed flask, and then 200 mL of distilled water were added. Then 10 mL of sodium ethylenediamine tetraacetate (EDTA) with a concentration of 10% (m/v) and 10 mL of phosphoric acid (C85% purity) were added before heating and mixing. Approximately 100 mL distilled solution containing cyanide from plant materials were collected, quantitatively transferred to a 100 mL volumetric flask, and made up to the volume with deionized water. The solution was stored below 6°C until the concentration of cyanide was determined. All samples were analyzed within a maximum holding time of 4 h. One to five milliliters of the aliquot solution samples were pippetted into a 25 mL colorimetric cylinder (depending on the concentrations of cyanide in solution), and 0.1% NaOH was added to make up the volume to 10 mL. Then 5.0 mL of a buffer solution with potassium dihydrogen phosphate and sodium phosphate were added.

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Quickly, 0.2 mL of 1% (m/v) chloramine-T solution were introduced. The vessel was sealed with a stopper and left standing for 3–5 min. Five mL of the colour reagent consisting of isonicotinic acid and 3-methyl-1-phenyl-5pyrazolone was then added. The content was diluted with deionized water to the mark (25 mL) and mixed thoroughly. Finally, the colorimetric cylinders were all kept in a water bath at a temperature of 32°C for 40 min. The absorption of light at 638 nm was measured in a cuvette of optical path length of 10 mm against water as a reference. All chemicals used were [ 99.5% purity, except potassium cyanide and nicotinic acid, which were technical grade (92–95% purity); but the stock solution and the standard solution of KCN used in this test were calibrated with a standard solution of AgNO3, which was also calibrated against a standard solution of NaCl (standard method of SEPA, P. R. China). The detection limit of this method was determined to be 0.004 mg CN/L, depending on the volume of the sample used. The sample preparation methods used in this study were also checked against samples spiked with certified solution standards and the mean recovery was 98.46%.

Phytovolatilization of Iron Cyanide Complexes Volatilization of iron cyanide complexes due to plant transpiration were measured using a modified test chamber. Treated plants were prepared as described above with the entire 50 mL Erlenmeyer flask–plant system enclosed in a glass chamber (10 9 10 9 30 cm) with air flowing through at 25°C. The tube at the outflow of the vessel was connected to a gas trap containing 5 mL of 1% sodium hydroxide to trap potentially airborne cyanide. The gas trap tube was wrapped with aluminum foil and changed daily, after which all gas tubes were analyzed for total cyanide. The duration of this test was 144 h. Statistical Methods Analysis of variance (ANOVA) and Tukey’s multiple comparison test were used to determine the statistical significance at 0.01 or 0.05 level between plant performances.

Results and Discussion Total Cyanide in Plant Materials Metabolic Response of Maize to Ferrocyanide The total cyanide in plant materials was analyzed based on the method by Yu et al. (2006). Plant materials from the treated and the untreated plants were harvested after 144 h of experiments. Fresh plant biomass cut into small pieces was used instead of 5 mL of the spiked sample. The remaining procedures were identical to those described before.

Selected physiological parameters of maize seedlings grown in the hydroponic solutions spiked with ferrocyanide were measured and compared with the controls (Table 2). A remarkable difference in selective parameters was found between the maize grown in the N-containing solution (S6)

Table 2 Transpiration rate, growth, soluble protein, and chlorophyll contents of maize exposed to ferrocyanide under different treatments. Values for transpiration and growth are the mean of nine replicates of both the treated and untreated control plants. Values for soluble protein and chlorophyll contents are the mean of three replicates, numerical values in brackets represent standard deviation, FW =

fresh weight. S1–5 refers to the treatment with different strength of N-free nutrient solution spiked with ferrocyanide. S6 refers to the treatment with 100% strength of N-containing nutrient solution spiked with ferrocyanide. Controls 1 and 2 refer to the treatments with 100% N-containing or N-free nutrient solution without addition of ferrocyanide

Characteristic

S1

S2

S3

S4

S5

S6

Control 1

Control 2

Transpiration rate (g /d)

1.67a (0.087)

1.61a (0.257)

1.56a (0.246)

1.51a (0.293)

1.55a (0.254)

2.01b (0.161)

1.98b (0.092)

1.58a (0.072)

Biomass growth (g)

0.68b (0.113)

0.68b (0.251)

0.38a (0.141)

0.37a (0.161)

0.41a (0.057)

0.75b (0.112)

0.76b (0.087)

0.48a (0.121)

Shoot growth (cm)

10.19(1.364)

10.61a (0.961)

9.29a (1.159)

8.45a (1.179)

7.91a (0.717)

15.76b (1.502)

15.56b (1.342)

9.89a (1.452)

Chlorophyll a (mg/g FW)

0.16a (0.027)

0.22a (0.025)

0.26a,b (0.008)

0.18a (0.013)

0.19a (0.018)

0.43b (0.050)

0.42b (0.021)

0.16a (0.028)

Chlorophyll b (mg/g FW)

0.16a (0.026)

0.21a (0.023)

0.25a,b (0.007)

0.18a (0.011)

0.18a (0.011)

0.43b (0.049)

0.42b (0.022)

0.16a (0.033)

Soluble protein (mg/g FW)

7.76a (0.567)

7.06a (0.824)

7.49a (0.291)

7.85a (0.824)

7.30a (1.271)

17.58b (1.873)

16.45b (1.654)

6.50a (0.545)

a

Significantly different to control 1 at a 95% significance level (two-tailed t-test)

b


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and those in the N-free solutions (S1–5) with ferrocyanide after 144 h exposure (P \ 0.01). When maize was planted in the N-containing nutrient solution amended with ferrocyanide, the selected physiological parameters were more or less identical to those grown in the same nutrient solutions without the test chemical (P [ 0.05), whereas significant decreases in the transpiration rate, biomass, shoot length, chlorophyll contents, and soluble proteins of maize seedlings grown in the N-free hydroponic solutions spiked with ferrocyanide were observed (P \ 0.01). A slight change in the selective parameters was also found with an increase of the strength of N-free nutrient solutions (P [ 0.05) implying that these physiological parameters of maize are susceptible to the availability of external nitrogen in the hydroponic solution than other nutrients. Although all monitored physiological parameters were significantly inhibited due to the lack of available nitrogen in the nutrient media, visible toxic symptom, e.g., chlorosis of leaves, was not observed in all treatment groups over the entire period of the study.

was detected for these parameters of maize seedlings grown in the N-free nutrient solutions regardless of ferricyanide addition (P \ 0.01). A minor effect of the strength of N-free nutrient solutions on the transpiration rate, biomass, shoot length, chlorophyll contents, and soluble proteins of maize was observed (P [ 0.05). These results collectively indicated that the physiological responses of maize seedlings are highly sensitive to the available nitrogen in the hydroponic solution, not ferricyanide. Visible symptoms of toxicity were not observed over the 144 h period of exposure.

Removal of Ferrocyanide from Hydroponic Solution by Maize

Table 3 shows the results of the responses of the selective physiological parameters of maize seedlings to ferricyanide. A negligible change was observed in the monitored parameters of maize grown in the N-containing solution spiked with ferricyanide compared with maize seedlings grown in the N-containing hydroponic solutions without ferricyanide as controls (P [ 0.05); a significant decrease

Figure 1 shows the measured concentrations of total cyanide in hydroponic solutions of different treatments after 144 h of exposure. A negligible change of the total cyanide concentration was observed in the aqueous solution of the controls without plants. Nearly all the applied ferrocyanide was removed from the N-free nutrient solutions in the presence of maize seedlings, whereas the total cyanide in the N-containing solution with maize seedlings was reduced from 10.14 to 8.24 (±1.381) mg CN/L after the 144-h of exposure. Between 94.4% and 96.6% of the amended ferrocyanide was removed from the N-free hydroponic solutions by the maize seedlings, while only 50.8% of the supplied ferrocyanide was removed from the N-containing hydroponic solution. This indicated that the additional nitrogen of inorganic form in the nutrient

Table 3 Transpiration rate, growth, soluble protein, and chlorophyll contents of maize exposed to ferricyanide under different treatments. Values for transpiration and growth are the mean of nine replicates of both the treated and untreated control plants. Values for soluble protein and chlorophyll contents are the mean of three replicates, numerical values in brackets represent standard deviation, FW =

fresh weight. S1–5 refers to the treatment with different strength of Nfree nutrient solution spiked with ferrocyanide. S6 refers to the treatment with 100% strength of N-containing nutrient solution spiked with ferrocyanide. Controls 1 and 2 refer to the treatments with 100% N-containing or N-free nutrient solution without the addition of ferricyanide

Characteristic

S1

S2

S3

S4

S5

S6

Control 1

Control 2

Transpiration rate (g/d)

1.50a (0.371)

1.49a (0.440)

1.48a (0.282)

1.45a (0.351)

1.55a (0.200)

2.20b (0.453)

2.16b (0.345)

1.58a (0.072)

Biomass growth (g)

0.60a (0.135)

0.56a (0.123)

0.54a (0.087)

0.58a (0.171)

0.65a (0.143)

0.83b (0.156)

0.82b (0.142)

0.48a (0.121)

Shoot growth (cm)

11.38a (1.552)

11.94a (1.985)

10.87a (1.414)

11.24a (1.521)

12.22a,b (2.433)

15.48b (2.282)

15.43b (1.675)

9.89a (1.452)

Chlorophyll a (mg/g FW)

0.20a (0.042)

0.19a (0.065)

0.18a (0.014)

0.21a (0.054)

0.19a (0.011)

0.47b (0.035)

0.45b (0.032)

0.16a (0.028)

Chlorophyll b (mg/g FW)

0.20a (0.043)

0.19a (0.060)

0.18a (0.013)

0.20a (0.051)

0.18a (0.012)

0.47b (0.036)

0.45b (0.031)

0.16a (0.033)

Soluble protein (mg/g FW)

5.56a (0.491)

5.33a (0.398)

5.64a (1.004)

6.53a (0.902)

6.79a (0.971)

14.57b (0.737)

14.87b (1.346)

6.50 (0.545)

Metabolic Response of Maize to Ferricyanide

a


b


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Fig. 1 Measured total cyanide concentration (mg CN/L) in the hydroponic solution spiked with ferrocyanide. The exposure period was 144 h. The values are the mean of three replicates for each sample. Vertical bars represent the standard deviation. I, initial concentration; F, final concentration

solution had a significant negative influence on the removal of ferrocyanide by maize seedlings (P \ 0.01). Figure 2 shows the concentrations of total cyanide in roots and shoots of maize seedlings grown in different treatment solutions after the 144 h of incubation. Cyanide was detected in plant materials in all treatment groups, confirming uptake and translocation of ferrocyanide by plants. The background cyanide concentration in non-treated maize was 0.069 (±0.021) mg CN/kg FW for roots (n = 3) and no cyanide was found above the detection limit in shoots of seedlings. Compared with the background, total cyanide in the roots and shoots of ferrocyanide-exposed maize seedlings was elevated and the difference among these treatment groups was not significant (P [ 0.05), but the total cyanide in roots was significantly higher than that in shoots for all treatments (P \ 0.01). After uptake from solution and translocation into shoots, ferrocyanide may be subjected to dissociation and then volatilization. However, no cyanide above the detection limit was trapped for maize seedlings exposed to ferrocyanide at 25°C over the 144 h of test period using an enclosed test chamber with gas trapping system. The same result was also found in a study conducted by Yu et al.

Fig. 2 Measured total cyanide concentration (mg CN/kg FW) in roots and shoots of maize exposed to ferrocyanide. The exposure period was 144 h. The values are the mean of three replicates for each sample. Vertical bars represent the standard deviation. FW, fresh weight

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(2006). Therefore, the mass balance of ferrocyanide was made from the residual total cyanide in solution and those recovered in plant materials (Table 4). Only 9.62–13.47% of the applied ferrocyanide was detected in the plant materials. Roots were the major sink for ferrocyanide accumulation and only trace amounts were recovered in shoots. Between 14.5% and 18.5% of the applied ferrocyanide were recovered from the hydroponic solutions and biomass of maize grown in the N-free solutions, whereas 63.3% was detected from the solutions and plant materials of maize grown in the N-containing solution. Therefore, all loss of ferrocyanide could be contributed to the assimilation and uptake by plant seedlings. This observation is consistent with several earlier findings (Ebbs et al. 2003; Yu et al. 2006; Larsen and Trapp 2006). The calculated assimilation rates of ferrocyanide are shown in Table 4. Rates of 19.63–28.40 mg CN/kg.d were found for maize seedlings grown in the N-free nutrient solutions. The difference in the assimilation rate among the different treatments was not significant (P [ 0.05). A significantly lower assimilation rate of ferrocyanide was obtained for maize grown in the N-containing solution with a value of 8.62 mg CN/kg.d (P \ 0.01). These results indicate that the presence of easily available nitrogen in plant growth media have a negative impact on the assimilation of ferrocyanide by maize.

Removal of Ferricyanide from Hydroponic Solution by Maize Figure 3 illustrates the changes of total cyanide concentrations in hydroponic solution spiked with ferricyanide. A negligible change of the total cyanide concentration in the aqueous solution of the controls without plant seedlings was observed. When maize was grown in the N-containing nutrient solution, the total cyanide in solution declined from 11.39 to 3.59 (±1.230) mg CN/L after the 144 hour of exposure, which accounted for 80.8% removal of the applied ferricyanide. When maize was incubated in the Nfree nutrient solutions spiked with ferricyanide, 61.3– 82.9% of the applied ferricyanide was removed from the hydroponic solution. The difference in the uptake of ferricyanide among the treatments with maize grown in the Nfree solution was significant (P \ 0.05), implying that lower strengths of N-free nutrient solution have a negative impact on the uptake of ferricyanide by maize seedlings. The total cyanide concentrations detected in plant materials of maize seedlings exposed to ferricyanide are presented in Fig. 4. Elevated cyanide concentrations were found in all parts of exposed maize, but a substantial difference was found in the distribution of cyanide in plant materials between the maize seedlings grown in the N-free


235

Table 4 Mass balance for plants exposed to ferrocyanide. Exposure period: 7 d; the values are the mean of three replicates; in brackets: standard deviation Treatment

S1 S2

Total cyanide in solution (lg)

Total cyanide in plant tissues (lg)

Initial

Final

Root

Shoot

562.50

25.63* (5.349)

64.16 (8.568)

0.69* (0.091)

523.00

*

26.27 (4.245)

70.23 (6.083)

0.21 (0.009) *

Total cyanide recovery (%)

Assimilation rate(mg CN/kg.d)

83.8* (2.100)

11.53 (1.570)

20.50* (3.256)

*

13.47 (3.068)

19.63* (0.573)

*

81.5 (3.823)

S3 S4

538.50 499.00

30.07 (5.281) 18.96* (4.758)

51.48 (5.822) 47.52 (4.959)

0.32 (0.058) 0.79* (0.125)

84.8 (4.761) 85.5* (2.959)

9.62 (2.914) 9.68 (2.482)

28.40* (6.622) 26.87* (4.768)

S5

419.00

16.92* (2.521)

61.78 (8.926)

0.74* (0.075)

83.8* (1.226)

12.73 (1.784)

21.55* (1.784)

S6

507.00

249.7 (14.98)

64.69 (6.431)

1.53 (0.248)

37.7 (2.746)

13.06 (3.109)

8.62 (1.399)

*

*

*

Total cyanide loss (%)

Significantly different to S6 at a 95% significance level (two-tailed t-test)

MðinitialÞ MðfinalÞ MðrootÞ MðshootÞ MðinitialÞ M þMðshootÞ Total cyanide recoveryð%Þ ¼ ðrootÞ 100 MðinitialÞ

Total cyanide lossð%Þ ¼

100

nutrient solutions and those grown in the N-containing nutrient solution. Total cyanide in the roots of maize grown in the N-free solutions was between 41.81 (±5.497) and 59.61 (±6.109) mg CN/kg FW and the variation among the five treatments was not significant (P [ 0.05), whereas a significantly higher concentration of total cyanide was found in the roots of maize grown in the N-containing nutrient solution with a value of 129.57 (±1.351) mg CN/

Fig. 3 Measured total cyanide concentration (mg CN/L) in the hydroponic solution spiked with ferricyanide at the beginning and end of the experiment. The exposure period was 144 h. The values are the mean of three replicates for each sample. Vertical bars represent the standard deviation

kg FW (P \ 0.01). Total cyanide in the shoots of the maize seedlings was detected between 0.45 (±0.130) and 0.64 (±0.202) mg CN/kg FW and no significant variation was detected among all the treatments (P [ 0.05). No cyanide above the detection limit was trapped in the gas trap due to plant transpiration. The mass balance of ferricyanide in maize is shown in Table 5. Only 7.89– 13.21% of the applied ferricyanide was recovered in the plant materials of maize grown in the N-free nutrient solutions, whereas 41.19% of the applied ferricyanide was detected in the biomass of the maize grown in the N-containing solution. Roots were the dominant sink for ferricyanide accumulation for all treatments and less than 1.0% of the ferricyanide accumulated in biomass was found in shoots. Between 83.7 % and 87.1 % of the ferricyanide removed from the N-free nutrient solutions were assimilated by maize at a rate of 23.53–27.47 mg CN/kg.d and the variation in the assimilation rate among the five treatments was not significant (P [ 0.05). More than 50% of the ferricyanide taken up from the solution remained in plant tissues of maize seedlings grown in the N-containing solution and a significantly lower assimilation rate of 9.42 mg CN/kg.d was obtained (P \ 0.01).

Discussion

Fig. 4 Measured total cyanide concentration (mg CN/kg FW) in roots and shoots of maize exposed to ferricyanide. The exposure period was 144 h. The values are the mean of three replicates for each sample. Vertical bars represent standard deviation. FW, fresh weight

Maize seedlings removed ferricyanide more effectively than ferrocyanide in the N-containing hydroponic solution (P \ 0.01). Interestingly, ferricyanide is not taken up readily, particularly as plants grown in the N-free hydroponic solutions generally take up the reduced form (Fe2+). These results indicate that the external nitrogen in the hydroponic solution had a significantly negative impact on the uptake of ferrocyanide by maize (P \ 0.01). Indeed, more than 95% of the applied ferrocyanide was removed by maize seedlings from the N-free hydroponic solutions,

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236


Table 5 Mass balance for plants exposed to ferricyanide. Exposure period: 7 d; the values are the mean of three replicates; in brackets: standard deviation Treatment

S1

Total cyanide in solution (lg)

Total cyanide in plant tissues (lg)

Initial

Final

Root

596.50

224.6* (17.36) *

Shoot

58.35* (6.736) *

0.37* (0.091) *

Total cyanide loss (%)

Total cyanide recovery (%)

52.5* (5.147)

9.84* (1.116)

23.53 (3.741)

*

Assimilation rate (mg CN/kg.d)

S2

589.50

228.0 (12.71)

49.90 (6.186)

0.48 (0.158)

52.8 (10.07)

8.55* (1.074)

26.93 (2.614)

S3 S4

644.00 549.50

236.2* (13.47) 93.92 (10.01)

50.43* (5.092) 60.46* (8.177)

0.37* (0.091) 0.37* (0.122)

55.4* (8.056) 71.8* (3.419)

7.89* (0.805) 11.07* (2.183)

27.47 (4.448) 24.92 (4.529)

S5

591.00

125.7 (18.32)

77.62* (7.801)

0.48 (0.158)

65.5* (7.136)

13.21* (1.313)

25.11 (2.435)

S6

569.50

109.6 (11.84)

233.7 (12.45)

0.90 (0.182)

39.6 (5.412)

41.19 (2.191)

9.42 (0.882)

*Significantly different to S6 at a 95% significance level (two-tailed t-test) MðinitialÞ MðfinalÞ MðrootÞ MðshootÞ MðinitialÞ M þMðshootÞ Total cyanide recoveryð%Þ ¼ ðrootÞ 100 MðinitialÞ

Total cyanide lossð%Þ ¼

100

whereas less than 51% was taken up from the N-containing solution. However, less applied ferricyanide was removed by maize grown in the N-free hydroponic solutions (B 60% strength) than those grown in the N-containing solution spiked with ferricyanide (P \ 0.05). As the strength of the N-free nutrient solutions increases, a minor difference in the removal rate of ferricyanide between the maize grown in the N-containing solution and those in the N-free solutions was observed (P [ 0.05). Additionally, higher removal rates of ferrocyanide were found in this study than other earlier findings (Ebbs et al. 2003; Samiotakis and Ebbs 2004; Larson and Trapp 2006; Yu et al. 2006), probably due to different species of plants used. Ferricyanide is purportedly membrane impermeable (Ebbs et al. 2003), therefore in vivo dissociation or biodegradation of ferricyanide to ferrocyanide or free cyanide would be a prerequisite before entering the plants. Plants assimilate iron through solubilization of Fe3+ by extracellular acidification, reduction of Fe3+ to Fe2+ by a plasma membrane redox system named ferrireductase first, uptake of Fe2+ can then be achieved by a specific transporter located in membrane (Marschner and Ro¨mheld 1994; Guerinot and Ying 1994; Eide 1997). This mechanism requires the obligatory reduction of extracellular Fe3+– chelate complexes before the splitting of the complex and then uptake of the released Fe2+. The conversion of ferricyanide to ferrocyanide or free cyanide is likely to occur prior to the uptake by plant roots. However, Larsen and Trapp (2006) observed that there was little support from their experimental results that ferricyanide was taken up as Fe2+ ion after reduction in solution. Additionally, in other previous findings willows showed a significantly higher removal capacity for free cyanide than iron cyanides (Ebbs et al. 2003; Yu et al. 2004; Larsen et al. 2005). In this study, although maize can efficiently remove both iron cyanide complexes, the removal rates between the two chemical forms are quite different, indicating that the conversion of

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ferricyanide to ferrocyanide or free cyanide in hydroponic solution prior to uptake by roots was unlikely to occur and different uptake mechanisms in maize exist. Results from mass balance analysis indicated that a significant difference in the accumulation of ferrocyanide in biomass between maize grown in the N-free hydroponic solutions and those in the N-containing nutrient solution (P \ 0.05). Indeed, 10.1–14.2% of the ferrocyanide taken up from the N-free hydroponic solution was recovered in the biomass of maize, whereas 25.6% of the ferrocyanide taken up was detected in plant materials of the maize grown in the N-containing nutrient solutions. Consequently, maize grown in the N-free hydroponic solution showed a significantly higher assimilation rate than those grown in the N-containing solution (P \ 0.01). A similar scenario was also observed in the treatment amended with ferricyanide. This indicated that the external nitrogen had a negative impact on the assimilation of both iron cyanide complexes in plant tissues. Interestingly, maize grown in the N-containing solution showed a significantly higher removal rate of ferricyanide than ferrocyanide (P \ 0.01), but a slight difference in the assimilation rate between the two chemical forms was obtained (P [ 0.05). This implied that the conversion of ferricyanide to ferrocyanide within plant tissues is most likely. It has been reported that free cyanide does not accumulate in healthy trees (Larsen et al. 2005). In this study, significantly higher concentrations of total cyanide in plant materials were found in roots and shoots of maize in all treatments in comparison with the background, implying that both iron cyanide complexes detected in plant tissues were probably still in the same original chemical complex forms. Larsen and Trapp (2006) also reported the same information. Additionally, the 15N was not detected in willow tissues as cyanide when 15N-labeled ferrocyanide was used (Ebbs et al. 2003). Therefore, we have a good reason to postulate that the assimilation pathways of free


cyanide and the iron cyanides are distinctively different and maize can efficiently assimilate both ferrocyanide and ferricyanide, especially when there is no other inorganic nitrogen available. Despite of the higher assimilation rates of both iron cyanides by maize grown in the N-free solutions, selective physiological parameters were significantly inhibited compared with the maize grown in N-containing nutrient solution. Therefore, neither of them can serve as a sole nitrogen source to support maize growth.

Conclusions The results of this laboratory study have demonstrated that maize was able to remove ferrocyanide and ferricyanide efficiently from N-free hydroponic solutions. Although no visible toxic symptoms were observed, selective physiological parameters of maize grown in the N-free solutions were significantly inhibited. The majority of the iron cyanides taken up from the N-free hydroponic solution was assimilated and small amounts were recovered in plant materials, mainly in the roots. Although maize grown in the N-containing nutrient solution showed a significantly higher removal rate of ferricyanide than ferrocyanide, similar assimilation rates were found. The results indicate that nitrogen from the plant nutrient solution has a strong negative influence on the uptake and assimilation of both iron cyanides. Results from this study suggest that uptake and assimilation mechanisms for ferrocyanide and ferricyanide may be different in maize and that the application of the external nitrogen has a substantial influence on the uptake and assimilation of both iron cyanides by plants. None of the iron cyanide complexes can serve as a nitrogen source to support maize growth. Acknowledgments This work was supported by a Ph.D. studentship from The University of Hong Kong. We thank Li-Qun Xing for his technical assistance and three anonymous reviewers for their comments in improving a previous version of this manuscript.

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