Journal of Ecology

Using

Plants

for the

Bioremediation (Phytoremediation) of ChromumContaminated Soils M.F. Abdel-Sabour* and Y.J. Al-Salama**

Manuscript ID: Manuscript Type:

Date Submitted by the Author: Complete List of Authors:

Key-words:

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Journal: draft n/a

Journal of Ecology

Standard Paper

Abdel-Sabour, mamdouh; Nuclear Research Center, Soil and Water Research Al-Salama, Yasser; 2nd Fac. Of Agric Aleppo Univ. Syria, Soil Sci. Dept. phytoremediation, , Heavy metal., Cr-uptake, accumulation, translocation, removal, Halophyte,

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Using Plants for the Bioremediation (Phytoremediation) of Chromum-Contaminated Soils

M.F. Abdel-Sabour and Y.J. Al-Salama
* **

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* Soil and water Dept, Nuclear Research Center,Egypt. *** Soil Sci. Dept. 2nd Fac. Of Agric (Der Ezzor) Aleppo Univ. Syria

Abstract

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A trial was made to study the use of hyperaccumulator plant species to extract Cr out of contaminated soils. Three soils (A, B and C) were selected in this experiment, Soil A: Polluted soil from El-Gabal El-Asfar farm. (subjected to sewage effluent irrigation for more than 75 years). Soil B: Polluted soil from Bahtem area (subjected to sewage effluent irrigation for more than 30 years). Soil C: Polluted soil from Mostorud area (irrigated with contaminated water for more than 30 years due to direct discharge of industrial wastewater to irrigation water canals). Four Kg of each air-dried surface soil sample (0-20cm) were packed in plastic containers in three replicates. Four plant species tested in this study namely, Sorghum (Sorghum Vulgar L.), Clover (Trifolium pratense L.), Panikum (Panicum antidotal) and Canola (Brasica Napous.); were grown on each tested soil in a complete randomized block experimental design. Plant shoots were harvested every 60 days (three cuts) for sorghum, clover and panikum. In case of canola plants, the shoots were harvested after 60 days (vegetative stage) and 85 days (fruiting stage). The roots of all species were collected after the final cut. Initial and final soil samples were taken for Cr analyses using neutron activation analyses technique (NAA).

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The cumulative biomass after three clippings showed that sorghum and canola exhibited the highest cumulative biomass. Canola accumulated the highest Cr uptake among the four plant species, irrespective of the clipping or soil type. Calculation of recovery percentage based on Cr removed from the soil after cultivation ranged between 3.7 to 40.6 % of total initial Cr. However, The percentage of Cr-removed by plant shoots from the initial total varied between 11

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to 17.9 % of the removed Cr, whereas the lowest values were observed in case of panikum and sorghum. The highest values were noticed in case of clover and Canola. The recovery percentage of Cr by root varied between 74 to 85.7 % of removed Cr from soil. This could be explained by the well-known fact that Cr is less mobile from roots to the shoots.

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Keywords: Bioremediation, , Heavy metal. Halophyte,

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There are several treatment technologies available for soils that have elevated levels of chromium. The technologies applicable to a particular chromium site depend on the clean-up goals, the form of the chromium present, and the volume and physical chemical properties of the chromium-containing soils. In many cases the clean-up goals are based on the Cr(VI) concentration in the soils. Therefore, most of the available treatment technologies consist of: (1) removing the Cr(VI)-containing soils from the site; (2) immobilizing the chromium so that it will not leach after treatment under field conditions; or (3) reducing the Cr(VI) in the soils to the Cr(III) state. Higgins et al., (1997) reviewed the available Cr-remediation strategies, their advantages and disadvantages, and their relative treatment costs. Technologies evaluated include excavation and offsite disposal, soil washing, soil flushing, electrokinetics,

solidification/stabilization, vitrification, and chemical and biological reduction. Particular emphasis is given to evaluating the technologies' ability to treat soils

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INTRUDUCTION

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that have Cr(VI) throughout the soil matrix rather than simply on the surface of the soil particles.

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A new research area of using plants for the bioremediation (phytoremediation) of contaminated soil and water was reviewed by Brown (1995). One of the earliest examples of a hyperaccumulator (plants that absorb metals in excess of their nutritional requirements without detrimental growth effects) was the Italian serpentine plant Alyssum bertolonii. another more recently identified hyper-accumulator is the alpine pennycress Thlaspi caerulescens. From Australia the tree Sebertia acuminata has a level of nickel sorption sufficient to turn its sap green and accumulate up to 20% dry wt is nickel (some species bleed blue sap due to chromium sorption). Most research into hyper-accumulators has concentrated on hydroponic systems whereas such metal uptake rates are greater than from soil where metal ions are less available through adsorption and complexation reactions. Problems associated with the application of plants to bioremediation problems are limited rooting depths, soil and environmental conditions not favourable to the test plant, differential accumulation and storage between the root and the shoot, restrictions to maximum plant mass, removal of metals through grazing by animals and insects, long duration of the remediation process, defining the allowable level of contamination, and problems of storage and disposal of resulting contaminated plant biomass.

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The aims of this study were to determine the potential of Cr-tolerant plants; and to determine the effect of phyto-extraction technique on Cr reduction on contaminated soils.

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MATERIAL AND METHODS Soil sampling: Three soil samples were chosen from different locations (three different locations at north greater Cairo, Egypt) to represent different soils (alluvial and sandy) as well as different sources of contaminated wastewater (sewage and industrial effluent). Soil A: Polluted soil from El-Gabal El-Asfar farm. (subjected to sewage effluent irrigation for more than 75 years). Soil B: Polluted soil from Bahtem area (subjected to sewage effluent irrigation for more than 30 years). Soil C: Polluted soil from Mostorud area (irrigated with contaminated water for more than 30 years due to direct discharge of industrial wastewater to irrigation water canals). Surface soil samples were collected. The samples were air-dried, crushed to pass a 2.0 mm sieve then soil properties were determined using the standard soil testing methods according to Jackson (1973). Available Cr was determined by DTPA method according to Lindsay and Norvell (1978), using atomic absorption spectrophotometer technique (AAS). Table (1) shows some physical and chemical properties of the tested soil samples. Phyto-extraction experiment: A trial was made to study the use of hyper-accumulator plant species to extract Cr out of contaminated soils. Three soils (A, B and C) were selected in this

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experiment, Four Kg of each air-dried surface soil sample (0-20cm) were packed in plastic containers (20) cm internal diameter and 20 cm in height) in three replicates. Four plant species tested in this study namely, Sorghum (Sorghum Vulgar L.), Clover (Trifolium pratense L.), Panikum (Panicum antidotal) and Canola (Brasica Napous.); were grown on each tested soil in a complete randomized block experimental design. Nitrogen and phosphorus fertilizer dose was applied to each soil before the cultivation of the plants at the recommended rates. Twenty Five seeds per pot of each tested plant species were planted. After 7 days, the plants were thinned to 15 plants/pot for sorghum, clover and panikum and 5 plants/pot for the canola specie. The soils were irrigated to maintain soil moisture at about 80 % of the soil field capacity during the 6 months period of the experiment. Plant shoots were harvested every 60 days (three cuts) by cutting the stems approximately 5cm above the soil surface for sorghum, clover and panikum. In case of canola plants, the shoots were harvested after 60 days (vegetative stage) and 85 days (fruiting stage). The roots of all species as well as all replicates were collected after the final cut. Initial and final soil samples were taken for Cr analyses.

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Plant samples (shoots and roots were dried weighed, dry ashed, digested (Chapman and Pratt, 1961) and analyzed for Cr concentration using (Atomic Adsorption Spectrophotometer Technique (AAS). Total Cr-soils (initial Cr and the residual in the soils after the experiment) was determined by Neutron Activation Analysis Technique (NAA). Three soil samples were analyzed for total Cr using Neutron Activation Analysis Technique

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(NAA). Soils samples were dried, ground and sieved to pass 500 mesh. An approximately 0.1 g of each soils sample was packed in pure Aluminum foil and prepared for NAA technique. A tenth grams of standard reference material soil (SL7) obtained from the International Atomic Energy Agency (IAEA) were capsuled as the soil samples. Moreover, an empty aluminum foil of known weight was irradiated on the same conditions, for identifying and subtracting the background -ray lines due to the aluminum envelopes. The capsules were

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irradiated in the second Egyptian reactor and subjected to neutron flux 1014 N/Cm2 /sec. for 48 hours. The samples were left to cool for 7 days. A full gamma spectrum for each sample was analyzed and calculated using a computerized multi-channel analyzer with high resolution by using hyperpure germanium detector ( resolution 1.9 KeV at 1332.5 KeV gamma-ray line of 60Co) associated with electronic circuits contains spectroscopy amplifier coupled to program system and an on-line multichannel computer The counting time of each sample was two hours and the dead time not exceeds 3 - 4 %. A computer code calculates the net area under the peak along the spectrum, which covers most of the trace elements. This area is used for calculating the concentration of Cr was detected using the relative methods according to the following equation (Ehmann and Vance, 1991):

Peak net area (sample) Concentration of element (sample) = Peak net area(standard) Concentration of element(standard)
Results and Discussion Dry matter accumulation:

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The mean dry matter (DM) yield from all clippings of sorghum, clover, and panikum (at 60 days intervals) and the two samples of canola (after 60 and 85 days) grown on tested soils (A,B and C) are shown in Table (2). The dry matter yield was significantly affected by soil factor, plant species and their interactions. For example, after 60 days the obtained dry matter yield could be arranged in the following descending order: Canola > Sorghum > Panikum> Clover. However, the cumulative biomass after three clippings showed that sorghum exhibited the highest cumulative biomass. Moreover, plants grown on fine texture soil exhibited the highest dry matter yield compared to those grown on coarse texture soil as shown in Table (2).

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Chromium phyto-Extraction:

The Cr content of plants has received much attention since the relatively recent discovery that Cr participates in glucose and cholesterol metabolism, and therefore is essential to man and animal (Ruhling and Tyler 1969). As shown in Table (3) canola accumulated the highest Cr uptake among the four plant species, irrespective of the clipping or soil type. Shoot concentrations of Cr were as high as 92 mg/kg dry matter of canola, followed by panikum and clover then sorghum with a range of Cr between 4 to 52 mg/kg dry matter. As shown in Table (3) the highest Cr uptake was observed in case of canola (either after 60 days or 85 days) compared with other plant species at any investigated soils. Then panikum showed higher Cr accumulation affinity compared to sorghum or clover especially in case of soil C.

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Soils total -Cr before and after cultivation are shown in Table (3). Calculation of recovery percentage based on Cr removed from the soil after cultivation (calculated on 4 kg soil bases) ranged between 3.7 to 40.6 % of total initial Cr. However, The percentage of Cr-removed by plant shoots from the initial total varied between 11 to 17.9 % of the removed Cr, whereas the lowest values were observed in case of panikum and sorghum. The highest values were noticed in case of clover and Canola. The recovery percentage of Cr by root varied between 74 to 85.7 % of removed Cr from soil. This could be explained by the well-known fact that Cr is less mobile from roots to the shoots. Most soils contain significant amounts of Cr, but its availability to plants is highly limited, and the rate of Cr uptake by plants is dependent on several soil and plant factors. Usually a higher Cr content is observed in roots than shoots. Phytotoxicity by Cr is seldom observed in the field, and specific accumulators of chromium are not known presently, though is several plants growing on serpentine soils or cultivated on tannery waste-amended soils, small increases in Cr concentrations have been found.

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Therefore, it was assumed that roots have a barrier effect that impedes or strongly limits Cr uptake and translocation from roots to shoots (Chaney et al., 1997). A low rate of Cr uptake by plant shoots from the soluble form is related to the mechanism of uptake by roots. These results agree with those obtained by Kabata-Pendias and Pendias, (1992). They reported that the Cr concentrations in plants vary widely for kinds of tissues and stages of growth. On the other hands, Tiffin (1977), concluded that Cr is

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transported in plants as anionic complexes which have been identified in the extracts of plants tissues and in xylem fluid. Table (2): Dry matter yield of different harvesting of tested plant species grown on contaminated soils. Plant species Sorghum LSD(0.05) Clover Soil Harvesting 60 days 85 days 120 days -80.6 A 94.8 -106.8 B 120.6 -125.2 C 141.0 ------44.2 57.6 68.0 39.2 43.6 52.3 ---180 days 77.2 93.2 115.2 60.1 70.2 78.2 35.2 42.1 49.3 ---Total * (g/pot) 252.6 320.6 381.4 3.97 137.6 169.0 205.1 1.89 118.6 142.5 172.8 2.82 116.2 137.2 205.6 1.49 2.45 2.35 4.07

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LSD(0.05) Pankium

LSD(0.05) Canola **

LSD(0.05) General LSD(0.05) Soil Plant Soil X Plant

*: Accumulative dry mater. **: Harvested After 60 and 85 days only.

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A 33.3 B 41.2 C 58.9 A 44.2 B 56.8 C 71.2 A 97.8 B 115.4 C 172.4 1.62 1.70 2.94

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116.2 137.2 205.6

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Conclusion The technologies applicable to a particular chromium site depend on the clean-up goals, the form of the chromium present, and the volume and physical chemical properties of the chromium-containing soils. In general, canola plant showed a higher affinity to remove soil-Cr by 23 to 40% of total soil Cr depending on soil-plant interaction. However, Cr-removal by plant roots was estimated to be often around 80% of total removed Cr.

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Brown, K.S. (1995), The green clean. BioScience. 1995, 45: 9, 579-582. Chaney, R.L.; J.A. Ryan and S.L. Brown (1997), Development of US_EPA limits for chromium in land-applied bio-solids and applicability of these limits to tannery by product derived fertilizers and other Cr-rich soil amendments. In S. Canali; F. Tittarell and P. Sepui (eds.) Chromium Environmental Issues. Milan: Agneli, pp. 229-295.

Chapman, H.D. and P.F. Pratt (1961), Methods of Analysis for Soils, Plant

and Waters. Berkeley Univ. California, USA.

Ehmann, W.D. and D.E. Vance (1991), Radiochemistry and Nuclear Method of Analysis. John Wiley and Sons, Inc., New York.

Higgins, T.E.; Halloran, A.R.; Petura,J.C.; Proctor,D.M; Finley,B.L.; Harris,M.A.; Paustenbach,D.J. and Rabbe,D. (1997) Traditional and innovative treatment methods for Cr(VI) in soil. J. Soil Contamination. 1997, 6: 6, 767-797. Jackson, M.L. (1973) Soil Chemical Analysis, Prentice Hall of India , Private limited, New Delhi.

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Referances

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Kabata-Pendias, A. and H. Pendias (1992), Trace Elements in Soil and Plant. 2nd Ed., CRC press, London. Lindsay, W. L. and W. A. Norvell (1978), Development of DTPA soil test for zinc, iron, manganese and copper. Soil Sci. Soc.. Am. J. 42:421-426. Ruhling, A. and G. Tyler (1969), Ecology of heavy metals: a regional and historical study. Bot. Not., 122:248. (C.F. Kabata-Pendias, A. and H. Pendias, 1992).

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Tiffin, L.O. (1977), The form and distribution of metals in plants: an overview, in Proc. Hanford Life Sci. Symp. U.S. Department of Energy, pp.315324.

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Table (1): Some physical and chemical properties of the experimental soils. Samples loction
El-Gabal EL-Asfar Soil A Bahtem Soil B Mostorud Soil C

Particle size distribution (%)

Coarse sand (2-0.2 mm)
Fine sand (0.2-0.02 mm) Silt (0.02-0.002 mm)

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58.02

21.81

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19.33 52.00 44.20

Clay (<0.002 mm)

Soil texture

pH

E.C.
mmhos/ cm.

CaCO3 (%)

O.M (%)

CEC meq/100g soil

5.67 17.62

21.53 13.87

20.80 24.31

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Sandy loam

6.91

1.23

0.70

6.17

13.26

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7.73 6.74

4.05 8.43

2.19 1.60

4.80 7.99

32.16 37.44

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Table (3): Recovery percentage of Cr removed from the tested soils by different plant species.

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Plant Species

Cr Initial (Total) (a) (mg/pot)

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After cultivation

(b) (mg/pot)

Sorghum 333.5 312.8 20.7 6.2 2.6 12.3 17.2 A 223.8 190.6 33.2 14.9 4.1 12.3 28.1 B 363.0 334.4 28.6 7.9 3.8 13.2 23.7 C Clover 333.5 321.2 12.3 3.7 1.7 13.5 9.1 A 223.8 204.6 19.2 8.6 3.5 17.9 14.4 B 363.0 339.4 23.6 6.5 3.7 15.8 18.4 C Panikum 333.5 291.9 41.6 12.5 4.6 11.0 35.7 A 223.8 174.9 48.9 21.9 5.6 11.4 41.9 B 363.0 313.0 50.0 13.8 5.5 11.0 42.2 C Canola 333.5 257.0 76.6 23.0 10.7 14.0 63.2 A 223.8 132.9 90.9 40.6 11.9 13.1 77.1 B 363.0 257.0 106.1 29.2 13.2 12.4 88.9 C * : Percentage of total removed from the initial metal in soil. **: Percentage removed by shoots or roots from the total remove.

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Cr Removed (a-b) (mg/pot)

Removal * (a-b/a)*100 (%)

Cr uptake by shoot (c) (mg/pot)

Removal ** by shoot
[c/(a-b)]*100

(%)

Cr uptake by root (d) (mg/pot)

Removal ** by root
[d/(a-b)]*100

(%)

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83.0 84.4 82.8 74.0 75.1 78.0 85.7 85.5 84.3 82.6 84.7 83.8

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