FOR PHYTOREMEDIATION OF SOILS ...

Journal of Environmental Protection and Ecology 18, No 2, 468–478 (2017) Soil pollution

POTENTIAL OF RAPESEED (Brassica napus L.) FOR PHYTOREMEDIATION OF SOILS CONTAMINATED WITH HEAVY METALS V. R. ANGELOVAa*, R. I. IVANOVAb, J. M. TODOROVb, K. I. IVANOVa Department of Chemistry, Agricultural University, Plovdiv, Bulgaria E-mail: [email protected] b Department of Plant Science, Agricultural University, Plovdiv, Bulgaria a

Abstract. A field study was conducted to evaluate the efficacy of rapeseed (Brassica napus L.) for phytoremediation of contaminated soils in the absence and presence of organic soil amendments (compost and vermicompost). The experiment was performed on an agricultural field contaminated by the Non-ferrous-metal Works near Plovdiv, Bulgaria. The field experimental was a randomised complete block design containing five treatments and four replications (20 plots). The treatments consisted of a control (no organic amendments), compost amendments (added at 20 and 40 t/da), and vemicompost amendments (added at 20 and 40 t/da). Upon reaching commercial ripeness, the rapeseed plants were gathered. The oil from ground seed was derived under laboratory conditions through an extraction method with the Socksle apparatus. Heavy metal contents in roots, stems, leaves, pods, seeds, oils and meals of rapeseed were determined. The addition of compost and vermicompost leads to decrease of the content of Pb, Zn and Cd, in the roots and stems, while the content of Pb and Cd increases in the pods of the rape. The addition of organic meliorants significantly reduces the content of the heavy metals of seeds and the oil. The oil from the control, as well as the oils obtained by processing the seeds from the variants with addition of compost and vermicompost, does not contain Cd, while Pb and Zn have values below the permissible limit values. The rape is a plant which is tolerant to heavy metals, can be grown in heavy metal polluted soils, and can be successfully used in the phytoremediation of heavy metal polluted soils. The possibility of further industrial processing of seeds to oil and using the obtained oil will make rapeseed economically interesting crops for farmers of phytoremediation technology. Keywords: phytoremediation, heavy metals, rapeseed, organic amendments.

AIMS AND BACKGROUND Heavy metal contamination of agricultural soils is a worldwide problem. The remediation of metal contaminated sites often involves expensive and environmentally invasive and civil engineering based practices. A range of technologies such as fixation, leaching, soil excavation, and landfill of the top contaminated soil ex situ have been used for the removal of metals. Many of these methods have high maintenance costs and may cause secondary pollution or adverse effect on biological activities, soil structure, and fertility1. Phytoremediation is an emerg*

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ing technology, which should be considered for remediation of contaminated sites because of its cost effectiveness, aesthetic advantages and long term applicability. This technology can be defined as the efficient use of plants to remove, detoxify or immobilize environmental contaminants in soils, waters or sediments through the natural, biological, chemical or physical activities and processes of the plants. It is best applied at the sites with shallow contamination of organic, nutrient or metal pollutants2. This plant-based technique is essentially an agronomic approach and its success depends ultimately on agronomic practices applied at the site. Addition of organic matter amendments, such as compost, fertilisers and wastes, is a common practice for immobilisation of heavy metals and soil amelioration of contaminated soils3. Organic amendments are able to improve soil physical, chemical and biological properties by: (i) raising the pH; (ii) increasing the organic matter content; (iii) adding essential nutrients for plant growth; (iv) increasing the water holding capacity, and (v) modifying heavy metals bioavailability4–6. The use of crop plants for phytoremediation of contaminated soils has the advantages of their high biomass production and adaptive capacity to variable environments7,8. However, to succeed they must be tolerant to the contaminants and be capable of accumulating significant concentrations of heavy metals in their tissues. Additionally, crops could make the long time-periods for decontamination more acceptable, economically and environmentally. If the contaminated biomass may be further proceed for added value products (not only concentrated on deposits of hazardous wastes), then such fact represents an improvement of economical efficiency of phytoremediation technology. Industrial plants, i.e. energy crops or crops for bio-diesel production, are therefore the prime candidates as plants for phytoremediation. The use of energy and/or bio-diesel crops as plants for phytoremediation would give contaminated soil a productive value and decrease remediation costs. Over the past years, there has been a growing interest in the use of oleaginous crops (such as rapeseed (Brassica napus)) for phytoextraction, or the sustainable use of land polluted with heavy metals9. Some plants of the Brassicaceae family have an extraordinary capacity to accumulate metals in their above-ground parts, some of which relate to the hyperaccumulators. For example, Thlaspi caerulescens can contain more than 10 000 mg/kg Zn in the leaves10. It has been found that within the genus Brassica, there are other types which have a tendency to accumulate high concentrations of metals and which can be characterised as metal accumulators. Examples include Brassica juncea (Indian mustard), Brassica rapa (field mustard) and Brassica napus (rape)11. It has been proven that Brassica juncea can accumulate high levels of heavy metals (Cd, Cr, Cu, Ni and Pb) when the solubility of the metals in the soil increases. Meers et al.12 found that Brassica rapa showed a high capacity for accumulation of Cd and Pb from the soil, with or without addition of supplements to mobilize elements from the soil. However, the accumulation is too 469

low, and the rapeseed is not suitable for phytoremediation. Marchiol et al.13 found that both Brassica species (Brassica napus and Raphanus sativus) are moderately tolerant when grown on soils slightly polluted with heavy metal. The main objective of this paper is to conduct a systematic study, which will help to determine the impact of organic soil amendments on the uptake of the heavy metals by rapeseed (Brassica napus L.), as well as the possibilities to use the plant for phytoremediation of heavy metal contaminated soils. EXPERIMENTAL The experiment was performed on an agricultural field contaminated by the Non-ferrous-Metal Works near Plovdiv, Bulgaria. The field experimental was a randomised complete block design containing five treatments and four replications (20 plots): 1 – introduction of 20 t/da of vermicompost to the soil, 2 – introduction of 40 t/da of vermicompost to the soil, 3 – introduction of 20 t/da of compost to the soil, 4 – introduction of 40 t/da of compost to the soil, 5 – control variant. Characteristics of soils and organic amendments are shown in Table 1. The soils used in this experiment were slightly acidic, with moderate content of organic matter and essential nutrients (N, P and K) (Table 1). The psedo-total content of Zn, Pb and Cd is high (1430.7 mg/kg Zn, 876.5 mg/kg Pb and 31.4 mg/kg Cd, respectively) and exceeds the maximum permissible concentrations (320 mg/kg Zn, 100 mg/kg Pb, 2.0 mg/kg Cd). Table 1. Characterisation of the soil and the organic amendments used in the experiment

Parameter pH EC (dS/m) Organic matter (%) N Kjeldal (%) C/N Pseudo-total P (mg/kg) Pseudo-total K (mg/kg) Pseudo-total Pb (mg/kg) Pseudo-total Zn (mg/kg) Pseudo-total Cd (mg/kg)

Soil    6.5    0.2    3.99    0.24    9.41  642 5518   876.5 1430.7   31.4

Compost     6.9     0.2    72.10     2.22    32.43 12654   6082    12.0    170.8     0.19

Vermicompost     7.5     2.2     38.58     1.57    24.59 10211 10495    32.3   270.3     0.69

To determine the effect of the organic amendments, the soil samples were collected 1 month after addition of organic amendments. A soil subsample was air-dried, passed through a 2-mm sieve and characterised for soil pH (H2O) in deionised water suspension of 1:5 (w/v); total nitrogen by the Kjeldahl method (N Kjeldahl); total oxidisable organic carbon according to Tube digestion method (with titration)14. 470

The pseudo-total and DTPA-extractable concentration of heavy metals in the soils, after four weeks equilibration were determined. Pseudo-total content of metals in soils was determined in accordance with ISO 11466 (Ref. 15). The available (mobile) heavy metals contents were extracted by a solution of DTPA (1 M NH4HCO3 and 0.005 M DTPA, pH 7.8) (Ref. 16). The same procedures were applied to organic amendments. The test plant was rapeseed (Brassica napus L.), hybrid Elit. Upon reaching commercial ripeness, the rapessed plants were gathered and the content of heavy metals in their different parts (roots, stems, pods and seeds) was quantitatively determined. The oil from rapeseed was derived under laboratory conditions through an extraction method with the Socksle apparatus, allowing the extraction of the oil from the preliminarily peeled and ground seeds of rapeseed by using petroleum ether and the subsequent liberation of the latter through distillation. The concentrations of Pb, Zn and Cd in their different parts (roots, stems and seeds), meals and oils were determined by the method of dry mineralisation. To determine the heavy metal content in the samples, inductively coupled emission spectrometer (Jobin Yvon Emission – JY 38 S, Paris, France) was used. Certified reference materials (contaminated brickworks soil – ERM CC135a and Apple leaves –1515 standard reference material) were used for quality control. The results show acceptable agreement between the found and certified values for Cd, Pb and Zn. Statistical analyses were conducted with Statistica v. 7.0. RESULTS AND DISCUSSION ACCUMULATION OF HEAVY METALS IN VEGETATIVE AND REPRODUCTIVE ORGANS OF RAPESEED WITHOUT AMENDMENT (CONTROL)

To clarify the issues of absorption, accumulation and distribution of heavy metals in vegetative and reproductive organs of rapeseed were analysed samples of roots, stems, pods and seeds. Table 2 presents the results obtained for the content of heavy metals in the vegetative and reproductive organs of the study oilseed crop. The content of Pb in the roots of rapeseed without amendments reached to 56.2 mg/kg, Zn – 330.70 mg/kg, Cd – 14.9 mg/kg. Our results indicate that a considerable part of the heavy metals are accumulated in the roots, which is consistent with the results of other authors17,18. This is explained by the fact that during the penetration of heavy metals in the plasma there is inactivation and disposal of significant quantities of them, as a result of the formation of slightly mobile compounds with the organic substance. According to Mathe-Gaspar and Anton19, rape accumulates mainly heavy metals in the roots, which is confirmed by our results. According to Turan and Esringu20, the content of Pb in the roots is 31 times higher than than in the aboveground mass. The high concentration of Cu and Pb in the roots and the low translocation factor indicate the possibility of rapeseed to be used in phyto471

stabilisation. Most of the heavy metals in the soil are fixed and accumulate in the roots of the rapeseed, as in rapeseed the main root mass is developed and is set in shallow in the surface soil horizon in which the heavy metal content is the highest. Perhaps this is the reason to accumulate a larger amount of heavy metals in the roots in the cultivation of rape on heavy metal polluted soils. Тable 2. Content of heavy metals (mg/kg) in vegetative and reproductive organs of rapeseed, oil and meal (without amendment, control)

Element Pb Cd Zn

Roots Stems Pods Seeds Oil Meal x ± sd x ± sd x ± sd x ± sd x ± sd x ± sd 56.2±2.5 9.65±0.8 66.1±3.6 4.3±0.2  0.05±0.005 6.9±0.2 14.87±1.3 11.85±0.5 10.5±2.1 1.0±0.1 nd 1.1±0.1 330.7±5.3 288.1±2.8 171.4±6.8 133.5±2.0 1.3±0.4 136.4±2.1

x – average value (mg/kg) from 5 repetitions; sd – mean standard deviation, nd – not detectable.

The content of Pb, Zn and Cd in stems of rapeseed is less compared to the root system, which indicates that their movement in the conduction system is severely limited. There is a minor accumulation of Pb in the stems of rape. The content of this element reaches 9.65 mg/kg in the stems, and is significantly lower than the toxic levels for the animals – 30 mg/kg (Ref. 21), despite that the total content of Pb in the soil from the field reaches up to 876.5 mg/kg and significantly exceeds the default values of 100 mg/kg for toxic effects on plants22. The results show moderate ability of rape to accumulate Cd in the aboveground mass. Such are the results of Ebbs and Kochian11 according to whom the content of Cd in the aboveground mass of B. napus reaches up to 3 mg/kg DW after a period of growth of 3 weeks in neutral clay soils with concentrations of total of Cd 40 mg/kg. The results are significantly lower than the values established by Ebbs and Kochian11 for Zn in the aerial aboveground mass of B. napus of 600 mg/kg in growing it for 3 weeks in neutral clay soils. The ratio of the heavy metals in the stem and the roots in the tested rape hybrid grown on heavy metal polluted soil under field conditions, is less than 1, which means that the hybrid is not an accumulator of heavy metals and can not be used for phytoextraction of heavy metals from the soil. Our results are in accordance with Rossi et al.23 and Kadar et al.24 according to whom the rapeseed accumulates moderate amount of heavy metals in the stems and has a relatively lower potential for phytoextraction compared to the wild species of the family Brassicaceae. The content of Pb, Cd and Zn in the fruits of the researched rape hybrid is higher than that of the stems. The higher content of heavy metals in the fruit shell is mainly due to their location and their rough surface, which is a prerequisite for possible anthropogenic pollution. Our results are consistent with those of Del Rio et al.25, according to whom significant amount of Pb and Zn is accumulated in the fruits of rapeseed. 472

The heavy metal content in the seeds was significantly lower compared to the root system and the aboveground mass of the plants. The content of Pb in the seeds of rape reaches up to 4.3 mg/kg, Zn up to 133.5 mg/kg and Cd up to 1.0 mg/kg. The results obtained strongly suggest that the pods act as a selective filter in the way of heavy metals to the seeds and depends primarily on the specificity of studied hybrid and the researched element. The statement of Dai et al.17 is confirmed according to which the content of Cd in the seeds of rape is very low, and the main part is localised in the carpellums of the fruits which appear the physiological barrier regarding the absorption of Cd. The results obtained are consistent with those of Korenovska and Palacekova26 and Dai et al.17, according to whom in the generative organs of rape grown on heavy metal polluted soils small amounts of Cu, Zn, Pb and Cd are accumulated. Significantly higher are the results of Grisspen et al.27, according to whom in the seed of rape the content of Cd varies between 3.6–8.1 mg/kg at a total content of Cd in the soil of 5.5 mg Cd/kg soil in experiments conducted in Belgium, and between 5.2 and 11.3 mg/kg at a total concentration of Cd in the soil 2.5 mg Cd/ kg soil in experiments conducted in the Netherlands. The results show that the major part of the heavy metals contained in the seeds, during processing do not pass into the oil, due to which their content is significantly lower. Pb content in rapeseed oil reaches up to 0.05 mg/kg and below the limit concentrations of Pb in vegetable oils (0.1 mg/kg) and it can be used for food purposes. Similar results were obtained for Zn and Cd. The values reported for Zn and Cd in rapeseed oil are respectively 1.3 and 0.02 mg/kg and lower than the maximum tolerances (10 mg/kg Zn and 0.05 mg/kg Cd). The comparative analysis between our results and those published in the literature shows that the content of Pb, Cd, and Zn is lower than the values of Przybyliski28, Darracq et al.29 and Farzin and Moassesi30, although rapeseed from our experiments was grown on heavily contaminated soil. The variation between the individual results may be due to growing conditions, genetic factors, varietal characteristics and other factors. Our results are in accordance with Anonymous31, according to whom the reported content of Cu, Fe and Pb in rape oil is low. The results obtained show that the main part of the heavy metals contained in the seeds during processing does not go into the oil, thus their content is significantly lower than the maximum permissible concentrations. The content of heavy metals in the rapeseed meal from control is shown in Table 2. Our results for Zn are higher in comparison with the published data (Bell32 – 72 mg/kg, Anonimous33 – 57–80 mg/kg), as rape is grown on heavy metal polluted soil. In terms of Pb and Cd we can not do a comparative analysis of our results as there is no data for these items published in the literature. The content of Pb in the control meals reaches up to 6.9 mg/kg, Zn – 136.4 mg/kg, and Cd – 1.08 mg/kg. The amounts of Pb and Zn in the meals are below the critical values 473

of 30 mg/kg Pb and 300 mg/kg Zn, while the content of Cd exceeds the critical value of 0.5 mg/kg Cd for feed. IMPACT ON ORGANIC AMENDMENTS ON ACCUMULATION OF HEAVY METALS IN THE VEGETATIVE AND REPRODUCTIVE ORGANS OF RAPESSED, RAPESEED OIL AND MEAL

The influence of compost on the uptake of heavy metals from the roots of rape is not unidirectional. The addition of 20 t/da of compost leads to increased content of Pb in the roots and stems of rape, while the addition of 40 t/da leads to decrease in the content of Pb, Zn and Cd in the roots and stems of rape (Fig. 1). The addition of compost leads to increase of the content of Pb and Cd in pods of rape, as this increase is more pronounced in the addition of 40 t/da of compost. The content of Zn in pods of rape decreases after the addition of compost, as this decrease is more pronounced after the addition of 40 t/da of compost. The addition of vermicompost also decreases the content of heavy metals in roots and stems compared to the control, as this decrease is more pronounced in the addition of 20 t/da vermicompost (Pb and Zn), and in the respect of Cd – in the addition of 40 t/ da of vermicompost (Fig. 1). Regarding the content of Pb and Cd in pods there is a tendency of increase, while in Zn a decrease is observed.

Fig. 1. Effect of different organic amendments (compost and vermicompost) applications to accumulation of heavy metals in the vegetative and reproductive organs of rapeseed

The decrease of the content of heavy metals in the seeds compared to the control is strongly expressed, as in the option with compost the Pb content is 474

reduced from 4.3 to 2.5 mg/kg, and in the option with vermicompost there is a slight increase (Fig. 1). Similar results are obtained for Cd. The Zn content in seeds of rape after the addition of organic meliorants decreases to a much lesser extent than Pb and Cd. The addition of compost and vermicompost reduces the content of Pb in the oil respectively to below the detection limits and these concentrations are lower than the maximum permissible concentrations of oil of plant origin (0.1 mg/kg) (Fig. 1). The addition of compost and vermicompost reduces Zn content in the oil, as this decrease is more strongly expressed in the addition of 40 t/da of compost and 40 t/da of vermicompost (corresponding to 0.97 and 1.08 mg/kg, respectively). In all variants, however, Zn content in the oil is lower than the maximum permissible concentrations of oil of plant origin (10 mg/kg). The content of Cd in rapeseed oil is below the limits of the quantitative measurement with the method used both in the control and in the the variants with the addition of organic meliorants. Rapeseed oil can be used for food purposes. The importation of compost and vermicompost leads to increased content of Pb in meals compared to the control of up to 7.58 mg/kg (40 t/da of compost) and 9.22 (20 t/da of vermicompost). Despite this increase in the Pb content, these values are lower than the maximum permitted concentrations for animal feed (30 mg/kg). The importation of 40 t/da of compost and 40 t/da of vermicompost reduces the content of Zn in meals, respectively to 109.53 and 96.33 mg/kg, and these values are lower than the maximum permitted concentrations of feed (500 mg/kg). The importation of organic meliorants leads to a slight increase in the content of Cd, with the exception of the option of deposit of 40 t/da of compost. The content of Cd in meals reduces to 0.975 mg/kg, but this concentration is higher than the maximum permissible levels for feed (0.5 mg/kg) (Ref. 34). The distribution of heavy metals in the organs of rape has a selective character and it is specific for the individual elements. The main part of Pb is accumulated in the pods (46%) and a very small amount is contained in the seeds (3%). With respect to Zn and Cd the main part is accumulated in the roots of rape (36 and 39%) followed by stems, pods and seeds. The seeds contain only 3% of the total amount of Cd absorbed by rape. Significantly higher is the content of Zn in the seeds – 14% of the total amount of absorbed Zn. The distribution of Pb in the organs of plants of all options with the addition of compost follows the same correlation observed in the control. The highest content of Pb was found in the pods, followed by the roots, stems and seeds. These results correspond to the results of Mathe-Gaspar and Anton19 who found that in the aboveground parts of the plants the accumulation of heavy metals is less than that in the roots.

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CONCLUSIONS It can be concluded thah the rape is a plant which is tolerant to heavy metals and can be grown in heavy metal polluted soils, and can be successfully used in the phytoremediation of heavy metal polluted soils. The addition of compost and vermicompost leads to decrease of the content of Pb, Zn and Cd, in the roots and stems, while the content of Pb and Cd increases in the pods of the rape. The addition of organic meliorants significantly reduces the content of the heavy metals of seeds and the oil. The oil from the control, as well as the oils obtained by processing the seeds from the variants with addition of compost and vermicompost, does not contain Cd, while Pb and Zn have values below the permissible limit values (0.1 mg/kg of Pb and 10.0 mg/kg of Zn, respectively). The processing of the seeds to oil and the use of the obtained oil will significantly reduce the cost for phytoremediation. REFERENCES  1. A. P. G. C. MARQUES, R. S. OLIVEIRA, A. P. G. C. RANGEL, P. M. L. CASTRO: Application of Manure and Compost to Contaminated Soils and Its Effect on Zinc Accumulation by Solanum nigrum Inoculated with Arbuscular Mycorrhizal Fungi. Environ Pollut, 151, 608 (2008). 2. X. E. YANG, H. Y. PENG, L. Y. JIANG: Phytoremediation of Copper from Contaminated Soil by Elsholtzia splendens as Affected by EDTA, Citric Acid, and Compost. International Journal of Phytoremediation, 7, 69 (2005). 3. R. CLEMENTE, M. P. WALKER, M. P. BERNAL: Uptake of Heavy Metals and As by Brassica juncea Grown in a Contaminated Soil in Aznalcóllar (Spain): the Effect of Soil Amendments. Environ Pollut, 136, 46 (2005). 4. D. J. WALKER, R. CLEMENTE, M. P. BERNAL: Contrasting Effects of Manure and Compost on Soil pH, Heavy Metal Availability and Growth of Chenopodium album L. in a Soil Contaminated by Pyritic Mine Waste. Chemosphere, 57, 215 (2004). 5. N. DRAGOMIR, S. MASU, C. BOGATU, B. LIXANDRU, M. LAZEROVICI, G. CRISTCA: On situ Remediation of Soils Polluted with Heavy Metals. Part I. Using of Supported Materials. J Environ Prot Ecol, 10 (1), 42 (2009). 6. S. MASU, V. RUS, M. ALBULESCU: Studies on Plant Growth and Metal Bioaccumulation in Crops on Fly Ash Disposal Site. J Environ Prot Ecol, 14 (3), 986 (2013). 7. M. KOMAREK, P. TLUSTOS, J. SZAKOVA, V. CHRASTNY, V. ETTLER: The Use of Maize and Poplar in Chelant-enhanced Phytoextraction of Lead from Contaminated Agricultural Soils. Chemosphere, 67,640 (2007). 8. A. FÄSSLER, B. H. ROBINSON, S. K. GUPTA, R. SCHULIN: Uptake and Allocation of Plant Nutrients and Cd in Maize, Sunflower and Tobacco Growing on Contaminated Soil and the Effect of Soil Conditioners under Field Conditions. Nutr Cycl Agroecosys, 87, 339 (2010). 9. L.van GINNEKEN, E. MEERS, R. GUISSON, A. RUTTENS, K. ELST, M. FILIP, M. G. TACK, J. VANGRONSVELD, L. DIELS, W. DEJONGHE: Phytoremediation for Heavy Metal‐contaminated Soils Combined with Bioenergy Production. J Environ Eng Landsc, 4, 227 (2007).

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