Environ Sci Pollut Res DOI 10.1007/s11356-016-6176-5
RESEARCH ARTICLE
Phytoextraction of Cd and Zn as single or mixed pollutants from soil by rape (Brassica napus) Paula Cojocaru 1 & Zygmunt Mariusz Gusiatin 2 & Igor Cretescu 3
Received: 1 September 2015 / Accepted: 25 January 2016 # Springer-Verlag Berlin Heidelberg 2016
Abstract This paper analyses the capacity of the rape (Brassica napus) to extract Cd and Zn from the soil and the effect of these metals on the morphometric parameters of the plant (length, weight, surface area, fractal dimension of leaves). Rape plants were mostly affected by the combined toxicity of the Cd and Zn mixture that caused a significant reduction in the rate of seed germination, the plant biomass quantity and the fractal dimension. In the case of Cd soil pollution, the bioaccumulation factor (BAF), bioaccumulation coefficient (BAC) as well as the heavy metal root-to-stalk translocation factor (TF) were determined. The results showed that B. napus had a great potential as a cadmium hyperaccumulator but not as an accumulator of Zn or Cd + Zn mixture. The efficiency of phytoextraction rape was 0.8– 1.22 % for a soil heavily polluted with cadmium.
Keywords Phytoremediation . Heavy metal pollution of soil . Bioaccumulation and translocation factor
Responsible editor: Elena Maestri * Igor Cretescu [email protected]
1
Faculty of Hydrotechnics, Geodesy and Environmental Engineering, BGheorghe Asachi^ Technical University of Iasi, 63-65 Blvd D. Mangeron, 700050 Iasi, Romania
2
Faculty of Environmental Sciences and Fisheries, Department of Environmental Biotechnology, University of Warmia and Mazury, ul. Sloneczna 45G, Olsztyn, Poland
3
Faculty of Chemical Engineering and Environmental Protection, BGheorghe Asachi^ Technical University of Iasi, 73 Blvd D. Mangeron, 700050 Iasi, Romania
Introduction Heavy metal soil contamination is one of the major global environmental issues, with significant risks for the human health as well as for ecosystems. The current decontamination technologies include soil excavation and soil washing followed by physical or chemical separation of contaminants (Prasad 2004). Unfortunately, such techniques are quite costly. However, a new and promising remediation strategy for decontamination of heavy metal-polluted soils is the phytoremediation process. Phytoremediation is a technology currently under development, which should be considered for decontaminating polluted sites due to its profitability, aesthetic advantages and long-term applicability (Malik et al. 2015; Surriya et al. 2015). Such technology can be defined as the effective use of plants in view of removing, detoxing or locking-in environmental contaminants in a growing matrix by means of natural, biological, chemical or physical actions and processes in plants (McCutcheon and Jørgensen 2008). The plant uptake efficiency depends on the soil type, plant species and metal mobility. Not all plants can be used in the phytoremediation process. The plants used must have the following features: high heavy metal tolerance, rapid growth, high biomass production, deep roots and capacity to uptake and transport metal from roots to stalks and leaves of the plant (Pirzadah et al. 2015). Many plants have been studied, but only a few meet the above-mentioned requirements. Plants like the Indian mustard (Brassica juncea), maize (Zea mays), pennycress (Thlaspi caerulescens), rice (Oryza sativa), sunflower (Helianthus annuus) or tall fescue (Festuca arundinacea) proved to have high tolerance to heavy metals; therefore, these have been used in phytoremediation studies (Rodríguez-Vila et al. 2015; Cang et al. 2011; Gheju and Stelescu 2013; Ubugunov et al. 2014; Liu et al. 2008; Murakami et al.
Environ Sci Pollut Res
2007; Mani et al. 2015; Marques et al. 2013; Caraiman Cojocaru and Macoveanu 2011; Xu and Wang 2013). However, further research is necessary to determine the most suitable species of plants. In this study, the rape (Brassica napus) was used for phytoremediation purposes. It belongs to the same family as the Indian mustard (Brassicaceae) and was used so far in order to establish phytotoxicity of mercury (Shiyab et al. 2009); phytoextraction of Cr, Cu, Pb and Zn (Brunetti et al. 2011); phytoextraction of Cd (Ehsan et al. 2014) and Pb by using citric acid (Shakoor et al. 2014); effects of Cu, Fe and Mn on the metabolism of plants (Jahangir et al. 2008); oxidative stress caused by copper excess (Russo et al. 2008) and the role of phytochelatins and antioxidants in the tolerance to Cd accumulation (Seth et al. 2008). Nevertheless, new studies are necessary to clarify and assess the phytoremediation capacity of such species of plants as regards the metals frequently occurring in soils, such as Cd and Zn. Cd was acknowledged as one of the most dangerous environmental contaminants. Cd occurrence in the soil most usually goes along with Zn occurrence. The negative effect of Cd on plants mainly implies breaking off atmospheric nitrogen synthesis processes, ammonification, nitrification and denitrification processes, blocking a large number of microbiological processes and decreasing the crops. Cd may also reduce the photosynthesis level and chlorophyll content; may induce oxidative stress and modify morphological, physiological or biochemical properties of various vegetal tissues (Kirkham 2006; Irfan et al. 2013). Zn is not as toxic as Cd, but its occurrence in soils in quantities exceeding natural levels may decrease microprocessing of organic matter by slowing down the fermentation process, may reduce the microorganisms’ activity in terms of cellulose decomposition and breathing processes, may decrease mass production of higher plants and cause a low level of toxicity in humans and animals that consume the plants cultivated on Zn-contaminated land plots (Broadley et al. 2007). Therefore, this study aims to determine the capacity of the rape (B. napus) to extract Cd and Zn from a soil polluted with
a single metal or a mixture of such metals. For this purpose, the Cd and Zn content was determined in the parts of B. napus plant (roots, stalks and leaves) as well as the effect of such metals on morphometric parameters (i.e. length, weight, surface area, fractal dimensions of leaves). The bioaccumulation factor (BAF), bioaccumulation coefficient (BAC) and the heavy metal root-to-stalk translocation factor (TF) were also calculated in the case of plants cultivated in a Cd-polluted soil. Rape plants used during the contaminant phytoextraction from Cd- and Zn-polluted soil will be incinerated, as a final processing step; these plants are not recommended for rapeseed oil extraction. The negative or positive effects of phytoextraction on rapeseed oil will make the object of a future studies.
Material and methods Experimental procedure The experiments were carried out in a greenhouse (the mean temperature ± SD = 25 ± 5 °C), located at the University of Warmia and Mazury in Olsztyn, Poland. The soil was collected from an arable land plot in Warmia and Mazury Province, Poland. The soil samples were air-dried and passed through a 5-mm sieve before analysis. The soil had a clay-sand texture (86 % sand, 11.8 % sediments, 2.2 % clay), 5.7 pH and low organic content (0.63 %). It contained 0.54 g/kg of N in total, 84.8 mg/kg of P, 76.4 mg/kg of Mg and 57.9 mg/kg of K. The soil was artificially contaminated with Cd and Zn, as single and mixed pollutants. The metals were added as a nitrate solution, generating the occurrence of divalent cations in soil. Two levels of metal concentration were studied. The experiments were conducted in seven variants, according to experimental data presented in Table 1. A precise amount of polluted soil (9 kg) was added in each experimental pot, thus obtaining a 5-L volume, where 25 rapeseeds (B. napus) were sown. The rapeseeds were provided by the Central Laboratory for Seeds and Propagating Material
Table 1 Experimental variants Experimental variants
Type of pollutant
Initial content of heavy metals in soil (mg/kg)
Number of repetition
I II III IV V VI VII
0 Zn Zn Cd Cd Zn + Cd Zn + Cd
0 335.14 1080.10 29.35 43.72 404.80 + 32.75 1002.26 + 60.10
4 4 4 4 4 4 4
Environ Sci Pollut Res Fig. 1 Effect of Cd and Zn pollution in soil on the biomass production of rape: a control, b soil polluted with Cd and c soil polluted with Zn
Quality, Bucharest, Romania. The rape plants were seedlings 4 days after sowing and were irrigated with a constant amount of water throughout the entire experiment. The plants’ height was measured during the growing period, at various time intervals. Two months after sowing, the rape plants were harvested and then separated into roots, stalks and leaves. The roots were washed with distilled water in order to remove any soil scraps. The roots, stalks and leaves were weighed by using a 0.1-μg precision balance. In addition, the length of separate parts of the plants, the surface area of leaves (single side without petiole) and fractal dimensions of leaves were measured by using digital techniques after scanning each analysed leaf (ImageJ 1.38 software). The roots, stalks and leaves of the biomass picked were dried in an oven at 65 °C for 24 h. The dried parts of the plants were crushed until a fine powder was obtained. Approximately 1.0 g of vegetal material was digested with con centrated nitric ac id in a micro wav e o ven (MARSXpress, CEM USA) using a one-stage microwave program (T = 170 °C, P = 800 W, t = 30 min). After cooling, the extracts were filtered by using Whatman 42 filter papers (8-μm pore size) into 25-mL glass flasks, which were filled up to their mark with distilled water.
Table 2
In order to measure total metal concentration in the soil, approximately 1.0 g of soil dried previously at 105 °C was digested in HCl:HNO3 mixture at 3:1 (v/v) ratio by using the same microwave program as in the case of plants. Each extract was filtered in 50-mL glass flasks. The metal concentrations in extracts were determined by means of a flame atomic absorption spectrometer (FAAS) (Varian, AA28OFS). The differences between the measured mean values of Cd and Zn content of each experimental variant were assessed by using ANOVA at significance value p < 0.05. Measuring morphometric parameters of plants Five of the most representative plants were selected from each pot after harvesting. To serve the study, photos of plants were taken and then the length of roots and stalks were measured by using ImageTool 3.0 software. The selected leaves were scanned at 300 DPI resolution, obtaining 8 bit bmp files. The total number of scanned leaves was 370. Any excedent (scanned spots, dust particles) was removed from the resulting images by using Paint software. The clean scans were then
The biomass of the plant separated into roots, stalks and leaves (values are given as means n = 3, ±standard deviation)
Type of pollutant
Content of heavy metals in soil (mg/kg)
Germination rate (%)
Mass of roots (g)
Mass of stalks (g)
Mass of leaves (g)
Total mass of plants (g)
0 Zn Zn Cd Cd Zn + Cd Zn + Cd
0 335.14 1080.10 29.35 43.72 404.80 + 32.75 1002.26 + 60.10
74 ± 2.31 33 ± 3.83 24 ± 8.64 92 ± 3.27 75 ± 3.83 40 ± 5.66 23 ± 6.83
14.85 ± 2.24 1.00 ± 0.22 0.75 ± 0.48 9.00 ± 2.57 1.85 ± 0.33 0.68 ± 0.35 0.65 ± 0.31
131.13 ± 11.76 1.58 ± 0.35 1.13 ± 0.41 21.18 ± 5.38 2.55 ± 0.82 1.15 ± 0.39 0.86 ± 0.30
119.88 ± 3.73 3.13 ± 0.30 1.50 ± 0.36 32.40 ± 8.38 6.30 ± 0.77 2.30 ± 0.51 1.38 ± 0.30
265.85 ± 17.73 5.70 ± 0.86 3.38 ± 1.25 62.58 ± 16.34 10.70 ± 1.92 4.13 ± 1.23 2.89 ± 0.91
Environ Sci Pollut Res Table 3
The morphometric parameters of rape (values are given as means n = 3, ±standard deviation)
Type of pollutant
Content of heavy metals in soil (mg/kg)
Parameters Roots
Stalks
Leaves
Length (mm)
Length (mm)
Length (mm)
Width (mm)
Area (cm2)
500.26 ± 1.86
501.00 ± 2.43
1083.35 ± 27.76
886.22 ± 2.63
89.21 ± 4.36
44.59 ± 1.38 29.47 ± 1.02
60.04 ± 0.22 33.65 ± 0.56
82.58 ± 5.27 50.94 ± 0.05
110.20 ± 8.13 67.91 ± 0.07
0.62 ± 0.24 0.17 ± 0.01
0
0
Zn Zn
335.14 1080.10
Cd
29.35
578.84 ± 1.62
459.87 ± 1.60
590.82 ± 19.03
488.49 ± 6.57
21.97 ± 2.22
Cd Zn + Cd
43.72 404.80 + 32.75
385.88 ± 1.45 106.07 ± 1.15
466.15 ± 1.34 145.57 ± 0.48
338.60 ± 7.13 58.39 ± 9.61
274.86 ± 7.05 74.29 ± 8.75
4.89 ± 0.62 0.26 ± 0.03
Zn + Cd
1002.26 + 60.10
77.52 ± 1.34
94.93 ± 0.34
41.01 ± 0.00
40.86 ± 0.00
0.19 ± 0.03
digitally analysed to obtain the measurements of leaves in terms of length, width (using ImageTool 3.0) and area (using ImageJ 1.38).
Results and discussion
Determination of the leaves fractal dimensions
The first negative effect of Cd and Zn on the rape (B. napus) was observed in the plants germination stage and later after the biomass production of plants. The biomass differences between experimental variants and control variant are presented in Fig. 1. The results obtained in terms of cadmium and zinc effects on the rape morphometric parameters for each experimental variant were compared with the reference sample, denominated variant I (corresponding to unpolluted soil). The mass of rape plants for each experimental variant is presented in Table 2. The largest amounts of biomass, including roots, stalks and leaves were obtained from a Cd-polluted
The box fractal dimension of each leaf was calculated by ImageJ 1.38 software using the same scanned image obtained for assessing the length, width and surface area of the leaf. ImageJ 1.38 software counts the number of boxes (Nb) necessary to cover the entire scanned object with different size grid boxes (d). The results were graphically represented as log (Nb) vs. log (d), and the fractal dimensions were calculated starting from the slope of the resulting line.
Fig. 2 Comparison of the forms of the rape leaves as a function of experimental variant: a control, b soil polluted with Cd, c soil polluted with Zn and d soil polluted with a mixture of Cd and Zn
The effect of Cd and Zn on the morphometric parameters of B. napus
Environ Sci Pollut Res
Fig. 5 Fractal dimension of leaves Brassica napus changes (p < 0.05) depending on Zn + Cd mixture concentration in soil Fig. 3 Fractal dimension of leaves Brassica napus changes (p < 0.05) depending on Cd concentration in soil
soil (23.54 %), whereas the lowest amounts were obtained from a soil polluted with a mixture of Zn and Cd (1.08 %). Comparing with the germination rate of the control sample the rapeseed germination was reduced down to 92 % or even down to 23 % under Cd or Cd and Zn mixture, respectively. The biomass of plants obtained decreased as the pollutant concentration increased. After increasing of Cd concentration in soil, from 29.35 to 43.72 mg/kg, the biomass of plants decreased by 53.27 %. Similarly, after increasing Zn concentration in the soil from 335.14 to 1080.10 mg/kg, the biomass of plants decreased by 59.30 %. Changes of morphometric features of plants are useful parameters to assess the negative impact of heavy metals. The values of such parameters are presented in Table 3. The toxicity effect was most visible in the leaves, especially in the presence of metal mixture
(96.22 %) at 1002.26 + 60.10 mg/kg concentration, followed by Zn at 1080.10 mg/kg concentration. As regards the soil pollution with Cd (43.72 mg/kg), the leaf length decreased by 68.75 %, which means that Zn toxicity was higher than Cd toxicity on leaves. A significant decrease of the root length was recorded for a Zn concentration of 1080.10 mg/kg (94.11 %), whereas for Zn + Cd mixture (1002.26 + 60.10 mg/kg), the decrease was lower (84.50 %) as compared to the unpolluted case (reference sample). In the case of Cd contamination, the root length decreased by only 22.87 % as compared to the unpolluted case (reference samples). The stalks were affected in the same way as the roots, and the most significant decrease was recorded for Zn at 1080.10 mg/kg (93.29 %) concentration.
The effect of Cd and Zn on leaves fractal dimensions
Fig. 4 Fractal dimension of leaves Brassica napus changes (p < 0.05) depending on Zn concentration in soil
Mandelbrot (1977) introduced the concept of fractal structure and dimensions. A fractal is an object with a comparable structure at different scales, and it is described as a parameter quantifying the structure of a complex object. Fractal means fragmented, irregular, fractional or discontinuous, and the fractal theory refers to forms defined by fundamental irregularity that develop regardless of the observation scale. Such theory is a method of interpreting nature, because the Euclidean geometry perceptions cannot adequately represent natural forms, according to Mandelbrot (1977). Fractals are self-similar forms; that means the structure of the entire system is frequently reflected in each fraction. Fractal objects are defined by the fractal dimensions (D), representing a number quantifying the irregularity and fragmentation level of a geometrical structure or natural object (Zmeskal et al. 2003). One of the most reliable and
43.72
Cd
330.16 350.41 620.69 715.91 505.10 1176.47
Roots Leaves Stalks Roots
The initial concentration of heavy metals in the soil (mg/kg)
335.14 1080.10 29.35
43.72 404.80 + 32.75 1002.26 + 60.10
Type of pollutant
Zn Zn Cd
Cd Zn + Cd Zn + Cd
2500.06 74.40 + 706.72 121.00 + 694.10
67.40 177.60 1134.88
Heavy metal concentration uptake by Brassica napus (mg/kg)
28.00
23.20
BAF
43.37 404.70 + 32.72 1002.24 + 59.20
335.13 1079.10 28.99
Heavy metal concentration in soil after phytoremediation (mg/kg)
Metal concentration in plant parts (mg/kg)
Leaves Stalks
Plant parts
Heavy metal concentrations in the rape biomass and phytoremediation efficiency
29.35
Cd
Table 5
The initial metal concentration in soil (mg/kg)
BAF, BAC and TF coefficients for Cd phytoextraction using Brassica napus
Type of pollutant
Table 4
26.90
21.14 16.37 11.55
11.25 11.94
BAC
0.80 0.0007 + 0.086 0.0005 + 0.048
0.001 0.0004 1.22
Phytoremediation efficiency (%)
1.04
1.09
TF
Environ Sci Pollut Res
Environ Sci Pollut Res
competitive methods of fractal analysis for a complex structure is the box-counting method. The box-counting method divides the Euclidean space in cubes of r length. Then, it counts the cubes containing points of the object (if the space considered is a plane, then the image is covered by squares). The fractal dimension of binary digital images Db, determined by box-counting method, measures the complex dimensions in the interval 1 ≤ Db ≤ 2 by calculating fractions between the increasing in detail and the scale (Zmeskal et al. 2001). The box-counting method is the most widely used to determine fractal dimensions, due to its precision and ease of implementation. That proved to be an effective instrument for describing, measuring and comparing natural features, from red blood cell aggregation (Oancea 2007) to forms of soils and geological rocks (Ai et al. 2014), as well as forms of plants and/or distributions (Du et al. 2013). The fractal dimension is also used as a toxicity indicator. Starting from such hypotheses, we intended to analyse fractal dimensions of rape leaves (B. napus) and study the shape modifications caused by toxicity of Cd and Zn, both used as single and mixed pollutants. The obtained results concerning the effect of cadmium and zinc on fractal dimensions of rape leaves for each experimental variant were compared with the unpolluted soil (reference sample), denominated experimental variant I. Thus, the fractal dimension values of rape leaves decreased as the soil pollutant concentration increased, depending on the type of metal. Figure 2 presents modifications of rape leaves caused by pollution with metals. Figures 3, 4 and 5 display fractal dimension values for rape leaves. From the obtained data, it came out that the lowest values of the fractal dimension were recorded for Zn + Cd mixture at a concentration of 1002.26 + 60.10 mg/kg (1.382). In the case of Zn, the fractal dimension values were 1.693 at a concentration of 335.14 mg/kg and 1.658 at a concentration of 1080.10 mg/kg, respectively. The highest fractal dimension values were recorded for the soil pollution with Cd: 1.847 at a soil concentration of 29.35 mg/kg and 1.727 at a soil concentration of 43.72 mg/kg. Such data confirm the higher toxicity effect of Zn on B. napus as compared to Cd. Phytoextraction of Cd by rape plants The phytoextraction of heavy metals using B. napus was assessed by means of three factors: BAF, BAC and TF, calculated by Eqs. 1–3 (Sekabira et al. 2011): . BA F ¼ C sh C s . BAC ¼ C p i C s . T F ¼ C sh C r
ð1Þ ð2Þ ð3Þ
where Csh and Cs are heavy metal concentrations from the aerial part of the plant (stalks) (mg/kg) and soil (mg/kg), Cp is the heavy metal concentration in the individual plant parts, i.e. (roots, stalks and leaves) (mg/kg), Csh and Cr are heavy metal concentrations in the stalk and the root, respectively (mg/kg). BAF is categorized as follows: 10 hyperaccumulator (Ma et al. 2001). Four categories of bioaccumulation coefficient (BAC) can be distinguished:
RESEARCH ARTICLE
Phytoextraction of Cd and Zn as single or mixed pollutants from soil by rape (Brassica napus) Paula Cojocaru 1 & Zygmunt Mariusz Gusiatin 2 & Igor Cretescu 3
Received: 1 September 2015 / Accepted: 25 January 2016 # Springer-Verlag Berlin Heidelberg 2016
Abstract This paper analyses the capacity of the rape (Brassica napus) to extract Cd and Zn from the soil and the effect of these metals on the morphometric parameters of the plant (length, weight, surface area, fractal dimension of leaves). Rape plants were mostly affected by the combined toxicity of the Cd and Zn mixture that caused a significant reduction in the rate of seed germination, the plant biomass quantity and the fractal dimension. In the case of Cd soil pollution, the bioaccumulation factor (BAF), bioaccumulation coefficient (BAC) as well as the heavy metal root-to-stalk translocation factor (TF) were determined. The results showed that B. napus had a great potential as a cadmium hyperaccumulator but not as an accumulator of Zn or Cd + Zn mixture. The efficiency of phytoextraction rape was 0.8– 1.22 % for a soil heavily polluted with cadmium.
Keywords Phytoremediation . Heavy metal pollution of soil . Bioaccumulation and translocation factor
Responsible editor: Elena Maestri * Igor Cretescu [email protected]
1
Faculty of Hydrotechnics, Geodesy and Environmental Engineering, BGheorghe Asachi^ Technical University of Iasi, 63-65 Blvd D. Mangeron, 700050 Iasi, Romania
2
Faculty of Environmental Sciences and Fisheries, Department of Environmental Biotechnology, University of Warmia and Mazury, ul. Sloneczna 45G, Olsztyn, Poland
3
Faculty of Chemical Engineering and Environmental Protection, BGheorghe Asachi^ Technical University of Iasi, 73 Blvd D. Mangeron, 700050 Iasi, Romania
Introduction Heavy metal soil contamination is one of the major global environmental issues, with significant risks for the human health as well as for ecosystems. The current decontamination technologies include soil excavation and soil washing followed by physical or chemical separation of contaminants (Prasad 2004). Unfortunately, such techniques are quite costly. However, a new and promising remediation strategy for decontamination of heavy metal-polluted soils is the phytoremediation process. Phytoremediation is a technology currently under development, which should be considered for decontaminating polluted sites due to its profitability, aesthetic advantages and long-term applicability (Malik et al. 2015; Surriya et al. 2015). Such technology can be defined as the effective use of plants in view of removing, detoxing or locking-in environmental contaminants in a growing matrix by means of natural, biological, chemical or physical actions and processes in plants (McCutcheon and Jørgensen 2008). The plant uptake efficiency depends on the soil type, plant species and metal mobility. Not all plants can be used in the phytoremediation process. The plants used must have the following features: high heavy metal tolerance, rapid growth, high biomass production, deep roots and capacity to uptake and transport metal from roots to stalks and leaves of the plant (Pirzadah et al. 2015). Many plants have been studied, but only a few meet the above-mentioned requirements. Plants like the Indian mustard (Brassica juncea), maize (Zea mays), pennycress (Thlaspi caerulescens), rice (Oryza sativa), sunflower (Helianthus annuus) or tall fescue (Festuca arundinacea) proved to have high tolerance to heavy metals; therefore, these have been used in phytoremediation studies (Rodríguez-Vila et al. 2015; Cang et al. 2011; Gheju and Stelescu 2013; Ubugunov et al. 2014; Liu et al. 2008; Murakami et al.
Environ Sci Pollut Res
2007; Mani et al. 2015; Marques et al. 2013; Caraiman Cojocaru and Macoveanu 2011; Xu and Wang 2013). However, further research is necessary to determine the most suitable species of plants. In this study, the rape (Brassica napus) was used for phytoremediation purposes. It belongs to the same family as the Indian mustard (Brassicaceae) and was used so far in order to establish phytotoxicity of mercury (Shiyab et al. 2009); phytoextraction of Cr, Cu, Pb and Zn (Brunetti et al. 2011); phytoextraction of Cd (Ehsan et al. 2014) and Pb by using citric acid (Shakoor et al. 2014); effects of Cu, Fe and Mn on the metabolism of plants (Jahangir et al. 2008); oxidative stress caused by copper excess (Russo et al. 2008) and the role of phytochelatins and antioxidants in the tolerance to Cd accumulation (Seth et al. 2008). Nevertheless, new studies are necessary to clarify and assess the phytoremediation capacity of such species of plants as regards the metals frequently occurring in soils, such as Cd and Zn. Cd was acknowledged as one of the most dangerous environmental contaminants. Cd occurrence in the soil most usually goes along with Zn occurrence. The negative effect of Cd on plants mainly implies breaking off atmospheric nitrogen synthesis processes, ammonification, nitrification and denitrification processes, blocking a large number of microbiological processes and decreasing the crops. Cd may also reduce the photosynthesis level and chlorophyll content; may induce oxidative stress and modify morphological, physiological or biochemical properties of various vegetal tissues (Kirkham 2006; Irfan et al. 2013). Zn is not as toxic as Cd, but its occurrence in soils in quantities exceeding natural levels may decrease microprocessing of organic matter by slowing down the fermentation process, may reduce the microorganisms’ activity in terms of cellulose decomposition and breathing processes, may decrease mass production of higher plants and cause a low level of toxicity in humans and animals that consume the plants cultivated on Zn-contaminated land plots (Broadley et al. 2007). Therefore, this study aims to determine the capacity of the rape (B. napus) to extract Cd and Zn from a soil polluted with
a single metal or a mixture of such metals. For this purpose, the Cd and Zn content was determined in the parts of B. napus plant (roots, stalks and leaves) as well as the effect of such metals on morphometric parameters (i.e. length, weight, surface area, fractal dimensions of leaves). The bioaccumulation factor (BAF), bioaccumulation coefficient (BAC) and the heavy metal root-to-stalk translocation factor (TF) were also calculated in the case of plants cultivated in a Cd-polluted soil. Rape plants used during the contaminant phytoextraction from Cd- and Zn-polluted soil will be incinerated, as a final processing step; these plants are not recommended for rapeseed oil extraction. The negative or positive effects of phytoextraction on rapeseed oil will make the object of a future studies.
Material and methods Experimental procedure The experiments were carried out in a greenhouse (the mean temperature ± SD = 25 ± 5 °C), located at the University of Warmia and Mazury in Olsztyn, Poland. The soil was collected from an arable land plot in Warmia and Mazury Province, Poland. The soil samples were air-dried and passed through a 5-mm sieve before analysis. The soil had a clay-sand texture (86 % sand, 11.8 % sediments, 2.2 % clay), 5.7 pH and low organic content (0.63 %). It contained 0.54 g/kg of N in total, 84.8 mg/kg of P, 76.4 mg/kg of Mg and 57.9 mg/kg of K. The soil was artificially contaminated with Cd and Zn, as single and mixed pollutants. The metals were added as a nitrate solution, generating the occurrence of divalent cations in soil. Two levels of metal concentration were studied. The experiments were conducted in seven variants, according to experimental data presented in Table 1. A precise amount of polluted soil (9 kg) was added in each experimental pot, thus obtaining a 5-L volume, where 25 rapeseeds (B. napus) were sown. The rapeseeds were provided by the Central Laboratory for Seeds and Propagating Material
Table 1 Experimental variants Experimental variants
Type of pollutant
Initial content of heavy metals in soil (mg/kg)
Number of repetition
I II III IV V VI VII
0 Zn Zn Cd Cd Zn + Cd Zn + Cd
0 335.14 1080.10 29.35 43.72 404.80 + 32.75 1002.26 + 60.10
4 4 4 4 4 4 4
Environ Sci Pollut Res Fig. 1 Effect of Cd and Zn pollution in soil on the biomass production of rape: a control, b soil polluted with Cd and c soil polluted with Zn
Quality, Bucharest, Romania. The rape plants were seedlings 4 days after sowing and were irrigated with a constant amount of water throughout the entire experiment. The plants’ height was measured during the growing period, at various time intervals. Two months after sowing, the rape plants were harvested and then separated into roots, stalks and leaves. The roots were washed with distilled water in order to remove any soil scraps. The roots, stalks and leaves were weighed by using a 0.1-μg precision balance. In addition, the length of separate parts of the plants, the surface area of leaves (single side without petiole) and fractal dimensions of leaves were measured by using digital techniques after scanning each analysed leaf (ImageJ 1.38 software). The roots, stalks and leaves of the biomass picked were dried in an oven at 65 °C for 24 h. The dried parts of the plants were crushed until a fine powder was obtained. Approximately 1.0 g of vegetal material was digested with con centrated nitric ac id in a micro wav e o ven (MARSXpress, CEM USA) using a one-stage microwave program (T = 170 °C, P = 800 W, t = 30 min). After cooling, the extracts were filtered by using Whatman 42 filter papers (8-μm pore size) into 25-mL glass flasks, which were filled up to their mark with distilled water.
Table 2
In order to measure total metal concentration in the soil, approximately 1.0 g of soil dried previously at 105 °C was digested in HCl:HNO3 mixture at 3:1 (v/v) ratio by using the same microwave program as in the case of plants. Each extract was filtered in 50-mL glass flasks. The metal concentrations in extracts were determined by means of a flame atomic absorption spectrometer (FAAS) (Varian, AA28OFS). The differences between the measured mean values of Cd and Zn content of each experimental variant were assessed by using ANOVA at significance value p < 0.05. Measuring morphometric parameters of plants Five of the most representative plants were selected from each pot after harvesting. To serve the study, photos of plants were taken and then the length of roots and stalks were measured by using ImageTool 3.0 software. The selected leaves were scanned at 300 DPI resolution, obtaining 8 bit bmp files. The total number of scanned leaves was 370. Any excedent (scanned spots, dust particles) was removed from the resulting images by using Paint software. The clean scans were then
The biomass of the plant separated into roots, stalks and leaves (values are given as means n = 3, ±standard deviation)
Type of pollutant
Content of heavy metals in soil (mg/kg)
Germination rate (%)
Mass of roots (g)
Mass of stalks (g)
Mass of leaves (g)
Total mass of plants (g)
0 Zn Zn Cd Cd Zn + Cd Zn + Cd
0 335.14 1080.10 29.35 43.72 404.80 + 32.75 1002.26 + 60.10
74 ± 2.31 33 ± 3.83 24 ± 8.64 92 ± 3.27 75 ± 3.83 40 ± 5.66 23 ± 6.83
14.85 ± 2.24 1.00 ± 0.22 0.75 ± 0.48 9.00 ± 2.57 1.85 ± 0.33 0.68 ± 0.35 0.65 ± 0.31
131.13 ± 11.76 1.58 ± 0.35 1.13 ± 0.41 21.18 ± 5.38 2.55 ± 0.82 1.15 ± 0.39 0.86 ± 0.30
119.88 ± 3.73 3.13 ± 0.30 1.50 ± 0.36 32.40 ± 8.38 6.30 ± 0.77 2.30 ± 0.51 1.38 ± 0.30
265.85 ± 17.73 5.70 ± 0.86 3.38 ± 1.25 62.58 ± 16.34 10.70 ± 1.92 4.13 ± 1.23 2.89 ± 0.91
Environ Sci Pollut Res Table 3
The morphometric parameters of rape (values are given as means n = 3, ±standard deviation)
Type of pollutant
Content of heavy metals in soil (mg/kg)
Parameters Roots
Stalks
Leaves
Length (mm)
Length (mm)
Length (mm)
Width (mm)
Area (cm2)
500.26 ± 1.86
501.00 ± 2.43
1083.35 ± 27.76
886.22 ± 2.63
89.21 ± 4.36
44.59 ± 1.38 29.47 ± 1.02
60.04 ± 0.22 33.65 ± 0.56
82.58 ± 5.27 50.94 ± 0.05
110.20 ± 8.13 67.91 ± 0.07
0.62 ± 0.24 0.17 ± 0.01
0
0
Zn Zn
335.14 1080.10
Cd
29.35
578.84 ± 1.62
459.87 ± 1.60
590.82 ± 19.03
488.49 ± 6.57
21.97 ± 2.22
Cd Zn + Cd
43.72 404.80 + 32.75
385.88 ± 1.45 106.07 ± 1.15
466.15 ± 1.34 145.57 ± 0.48
338.60 ± 7.13 58.39 ± 9.61
274.86 ± 7.05 74.29 ± 8.75
4.89 ± 0.62 0.26 ± 0.03
Zn + Cd
1002.26 + 60.10
77.52 ± 1.34
94.93 ± 0.34
41.01 ± 0.00
40.86 ± 0.00
0.19 ± 0.03
digitally analysed to obtain the measurements of leaves in terms of length, width (using ImageTool 3.0) and area (using ImageJ 1.38).
Results and discussion
Determination of the leaves fractal dimensions
The first negative effect of Cd and Zn on the rape (B. napus) was observed in the plants germination stage and later after the biomass production of plants. The biomass differences between experimental variants and control variant are presented in Fig. 1. The results obtained in terms of cadmium and zinc effects on the rape morphometric parameters for each experimental variant were compared with the reference sample, denominated variant I (corresponding to unpolluted soil). The mass of rape plants for each experimental variant is presented in Table 2. The largest amounts of biomass, including roots, stalks and leaves were obtained from a Cd-polluted
The box fractal dimension of each leaf was calculated by ImageJ 1.38 software using the same scanned image obtained for assessing the length, width and surface area of the leaf. ImageJ 1.38 software counts the number of boxes (Nb) necessary to cover the entire scanned object with different size grid boxes (d). The results were graphically represented as log (Nb) vs. log (d), and the fractal dimensions were calculated starting from the slope of the resulting line.
Fig. 2 Comparison of the forms of the rape leaves as a function of experimental variant: a control, b soil polluted with Cd, c soil polluted with Zn and d soil polluted with a mixture of Cd and Zn
The effect of Cd and Zn on the morphometric parameters of B. napus
Environ Sci Pollut Res
Fig. 5 Fractal dimension of leaves Brassica napus changes (p < 0.05) depending on Zn + Cd mixture concentration in soil Fig. 3 Fractal dimension of leaves Brassica napus changes (p < 0.05) depending on Cd concentration in soil
soil (23.54 %), whereas the lowest amounts were obtained from a soil polluted with a mixture of Zn and Cd (1.08 %). Comparing with the germination rate of the control sample the rapeseed germination was reduced down to 92 % or even down to 23 % under Cd or Cd and Zn mixture, respectively. The biomass of plants obtained decreased as the pollutant concentration increased. After increasing of Cd concentration in soil, from 29.35 to 43.72 mg/kg, the biomass of plants decreased by 53.27 %. Similarly, after increasing Zn concentration in the soil from 335.14 to 1080.10 mg/kg, the biomass of plants decreased by 59.30 %. Changes of morphometric features of plants are useful parameters to assess the negative impact of heavy metals. The values of such parameters are presented in Table 3. The toxicity effect was most visible in the leaves, especially in the presence of metal mixture
(96.22 %) at 1002.26 + 60.10 mg/kg concentration, followed by Zn at 1080.10 mg/kg concentration. As regards the soil pollution with Cd (43.72 mg/kg), the leaf length decreased by 68.75 %, which means that Zn toxicity was higher than Cd toxicity on leaves. A significant decrease of the root length was recorded for a Zn concentration of 1080.10 mg/kg (94.11 %), whereas for Zn + Cd mixture (1002.26 + 60.10 mg/kg), the decrease was lower (84.50 %) as compared to the unpolluted case (reference sample). In the case of Cd contamination, the root length decreased by only 22.87 % as compared to the unpolluted case (reference samples). The stalks were affected in the same way as the roots, and the most significant decrease was recorded for Zn at 1080.10 mg/kg (93.29 %) concentration.
The effect of Cd and Zn on leaves fractal dimensions
Fig. 4 Fractal dimension of leaves Brassica napus changes (p < 0.05) depending on Zn concentration in soil
Mandelbrot (1977) introduced the concept of fractal structure and dimensions. A fractal is an object with a comparable structure at different scales, and it is described as a parameter quantifying the structure of a complex object. Fractal means fragmented, irregular, fractional or discontinuous, and the fractal theory refers to forms defined by fundamental irregularity that develop regardless of the observation scale. Such theory is a method of interpreting nature, because the Euclidean geometry perceptions cannot adequately represent natural forms, according to Mandelbrot (1977). Fractals are self-similar forms; that means the structure of the entire system is frequently reflected in each fraction. Fractal objects are defined by the fractal dimensions (D), representing a number quantifying the irregularity and fragmentation level of a geometrical structure or natural object (Zmeskal et al. 2003). One of the most reliable and
43.72
Cd
330.16 350.41 620.69 715.91 505.10 1176.47
Roots Leaves Stalks Roots
The initial concentration of heavy metals in the soil (mg/kg)
335.14 1080.10 29.35
43.72 404.80 + 32.75 1002.26 + 60.10
Type of pollutant
Zn Zn Cd
Cd Zn + Cd Zn + Cd
2500.06 74.40 + 706.72 121.00 + 694.10
67.40 177.60 1134.88
Heavy metal concentration uptake by Brassica napus (mg/kg)
28.00
23.20
BAF
43.37 404.70 + 32.72 1002.24 + 59.20
335.13 1079.10 28.99
Heavy metal concentration in soil after phytoremediation (mg/kg)
Metal concentration in plant parts (mg/kg)
Leaves Stalks
Plant parts
Heavy metal concentrations in the rape biomass and phytoremediation efficiency
29.35
Cd
Table 5
The initial metal concentration in soil (mg/kg)
BAF, BAC and TF coefficients for Cd phytoextraction using Brassica napus
Type of pollutant
Table 4
26.90
21.14 16.37 11.55
11.25 11.94
BAC
0.80 0.0007 + 0.086 0.0005 + 0.048
0.001 0.0004 1.22
Phytoremediation efficiency (%)
1.04
1.09
TF
Environ Sci Pollut Res
Environ Sci Pollut Res
competitive methods of fractal analysis for a complex structure is the box-counting method. The box-counting method divides the Euclidean space in cubes of r length. Then, it counts the cubes containing points of the object (if the space considered is a plane, then the image is covered by squares). The fractal dimension of binary digital images Db, determined by box-counting method, measures the complex dimensions in the interval 1 ≤ Db ≤ 2 by calculating fractions between the increasing in detail and the scale (Zmeskal et al. 2001). The box-counting method is the most widely used to determine fractal dimensions, due to its precision and ease of implementation. That proved to be an effective instrument for describing, measuring and comparing natural features, from red blood cell aggregation (Oancea 2007) to forms of soils and geological rocks (Ai et al. 2014), as well as forms of plants and/or distributions (Du et al. 2013). The fractal dimension is also used as a toxicity indicator. Starting from such hypotheses, we intended to analyse fractal dimensions of rape leaves (B. napus) and study the shape modifications caused by toxicity of Cd and Zn, both used as single and mixed pollutants. The obtained results concerning the effect of cadmium and zinc on fractal dimensions of rape leaves for each experimental variant were compared with the unpolluted soil (reference sample), denominated experimental variant I. Thus, the fractal dimension values of rape leaves decreased as the soil pollutant concentration increased, depending on the type of metal. Figure 2 presents modifications of rape leaves caused by pollution with metals. Figures 3, 4 and 5 display fractal dimension values for rape leaves. From the obtained data, it came out that the lowest values of the fractal dimension were recorded for Zn + Cd mixture at a concentration of 1002.26 + 60.10 mg/kg (1.382). In the case of Zn, the fractal dimension values were 1.693 at a concentration of 335.14 mg/kg and 1.658 at a concentration of 1080.10 mg/kg, respectively. The highest fractal dimension values were recorded for the soil pollution with Cd: 1.847 at a soil concentration of 29.35 mg/kg and 1.727 at a soil concentration of 43.72 mg/kg. Such data confirm the higher toxicity effect of Zn on B. napus as compared to Cd. Phytoextraction of Cd by rape plants The phytoextraction of heavy metals using B. napus was assessed by means of three factors: BAF, BAC and TF, calculated by Eqs. 1–3 (Sekabira et al. 2011): . BA F ¼ C sh C s . BAC ¼ C p i C s . T F ¼ C sh C r
ð1Þ ð2Þ ð3Þ
where Csh and Cs are heavy metal concentrations from the aerial part of the plant (stalks) (mg/kg) and soil (mg/kg), Cp is the heavy metal concentration in the individual plant parts, i.e. (roots, stalks and leaves) (mg/kg), Csh and Cr are heavy metal concentrations in the stalk and the root, respectively (mg/kg). BAF is categorized as follows: 10 hyperaccumulator (Ma et al. 2001). Four categories of bioaccumulation coefficient (BAC) can be distinguished:
![3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus).](/assets/img/hold.png)
