Fresenius Envir Bull 9: 328 - 332 (2000) ~ Editors, Freising-WeihenstephanlFRG 1018-4619/1000/S-6/328-0S DM 20 or 3,SO/p

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FIELD TRIALS OF BRASSICA CARINATA ~ND BRASSICA JUNCEA IN POLLUTED SOll-S OF THE GUADm.:MAR RIVER AREA.
M. del Ro*, R. Font*, 1. Fernndez-Mart~nez*, 1. Domnguez** and A. de Haro*. * Institute of Sustainable Agriculture, ICSlc. Av. Alameda del Obispo s/n. ** CIFA Av. Alameda del Obis~o s/n E-1480. Crdoba. Spain.

SUMl\'IARy Phytoextraction is a subjet of phytoremediation 'n which metal-accumulating plants are used to transport and concentrate metals from soils joto he harvestable parts1. In this study we test the capacity of some genotypes of Brassica juncea L. Czern. & Coss.) and Brassica carinata (A. Braun) for uptaking heavy metals from polluted oils after the toxic spill of the Aznalcollar mine (southern Spain). We present the levels of P , Zn, Cu and Cd found in the soil and the concentrations ofPb, Zn and Cu in the harvested p ans. High concentrations ofPb, Zn and Cu were present in the soil. B. juncea had greater eapacit for uptaking than B. carinata for all the metals analysed except for Cu. Key words: heavy metals, uptake, Brassica carina~a,Brassicajuncea, phytoremediation

INTRODUCTION The toxic spill of the Aznalcllar mine on APril 2S, 1998, in the proximity of a majar wild life reserve such as the Doana National Park (south m Spain) eaused a discharge of aeid waters and
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pyritie slurry (S 106 m3) to the Guadiamar river a d adjacent agricultural areas (SOOO Ha.). Due to this soils have remained polluted by heavy metalslsuch as Pb, Cu, Zn, Cd, TI, Sb and metalloids as As.
Recently, phy10remediation has emerged as an aIt~rnative to the engineering based methods. In this new approach plants are used to uptake eontamin~nts freID the soil and to transIoeate them to the shoots. Pollutants are then removed by harvestin~ the aboveground tissue for subsequent volume reduction (i.e. ashing) and storage. Phytoreme4iation is a eost-effective technique that could

remediate a site without dramatically disturbing thF landseape. A small number of plant species have been identi~ed that are not only eapable of growing on soils
eontaining high levels of met,als but also aeeumul~ti~g those polluta~ts ~o high e~ncentrations in the shoots. These pIants were comed hyperaccumuIat~rs-. On sueh spee1es 1S Thlaspz caerulescens L, a Presented at the 10thIntemational Symposium o~MESAEP in Alicante, Spain, 2 - 6 Oet. 1999

M. del Rio et al. member of the Brassicaceae family and a well known Z~ and Cd hyperaccumulator3.

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The success of any phytoremediation technique depends upon the identification of suitable plant species that hyperaccumulate heavy metals and produce arge amounts ofbiomass using established crop production and management practices.
r

Recent evidence has suggested that other species from fhe Brassicaceae family, particularly those ITomthe genus Brassica, may be more effective. specir such as Brassica juncea, Brassica napus L and Brassica rapa L, have been shown to accumulate moderate levels of heavy metals4-5. B. juncea and B. carinata were chosen in our study bec4use they are well adapted to mediterranean climate and they produce at least 20 times more b~omass iban T. caerulencens under field conditions6. The specific objective of ibis experiment was to dettrmine the suitability of B. juncea and B. carinata as phytoextraction species based on its growth responses and heavymetal uptake when grown on contaminated soil.

MA TERIAL A1'j) METHODS

B. carinata and B. juncea were the species selected tJ_~onduct this study. The sowing was made handly on March 15, 1999. The distance between rors was 50 cm with 50 seeds/linear meter. Plants were harvested on June 18, 1999. At ibis time, the plants had low biomass and seed yield due to the late sowing and lack of rain during the growing Pfriod. The levels of heavy metals in the soil were estimated *y Departament of Edafology and Chemical Agriculture of the University of Granada (Spain). T ' e samples were digested with nitric and
hydrochloric acids and analysed fo, Pb. Zn. Cu and Cd sing an inductively coupled plasma (ICP) spectrometer (Perkin Elmer Model SCIEX-Elan-5000A .

All plants were divided into leaves, seeds, pods, ste1s and reGs. The samples were thoroughly washed with tap water and given a final rinsing with delonized water and then dried at 80C (degree celsius) for 48 hours in an oyen and ground in a mili, ,nalysing them separately for Pb, Zn, Cu and Cd. To prepare the samples for heavy metals analysis,ldry material (ca 250 mg) was digested with 3mI..of nitric acid in a conical flask at a temperature o 13O0C and then with 1ml. of perchloric acid at 230C. After cooling H2O was added to the acid sol tion until a volume of 15m!. Pb, Zn, Cu, Fe and Mn were determined using flame by atomic abs; rption spectrometry (perkin Elmer. 1100B) I and expressed as mg Kg-1dry weight ofplant tissue.

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I RESULTS AND DISCUSSION

M. del Ro et al.

The results obtained showed that the soil anal sed contained low concentraran of Cd and high

j hough plant available metal was low due to the concentrations of Pb, Zn and Cu (Table 1), al
previous chemical treatments (soi! amendments ~ith calcium carbonato and ferric oxides) used to

fix metals in the soil.

Table 1. Total, lant available metal and soluble ea metal concentrations in soil. Heavy metal Horizons Total metal Available metal Soluble metal cm
Pb 1 2 3 1 2 3 1 2 3 309,57 258,06 30,64 461,72 393,43 64,24 127,97 107,30 20,93 mg Kg-I dry soil 14,13 14,86 5,27 114,71 130,56 13,37 32.53 28.52 1,45 0,30 0,27 0,70 0,55 0,27 0.38 0.21

Zn

Cu

Cd

1 2 3

1,51 1,27 0,12

15.24 1,20 0,88 0,09

0.09 0,01 0,007 0,002

1, 2, 3 are horizons

1-10 cm. 10-30 cm and >30 cm re'pe

l.ve1Y.

The polluted soil showed a gradient of concentrrtion for all the metals analysed, decreasing the values freIDthe first horizon to the third. The total content of metal found in the three hon ons ranged 30.64 to 309.57 mgKg-1for Pb; 64.24 to 461.72 rngKg-1for Zn; 20.93 to 127.97 mgKg-1 or Cu and 0.12 to 1.51 rngKg-1for Cd. Plants of B. juncea accumulated higher amounts t an B. carinata for all the metals analysed except for Cu (Table 2), as has been previously reported4.
Table 2. Concentrations ofPb, Zn and Cu in BrafiCajunCea Species Pb Zn and Brassica carinata plants. Results Cn

are means of 5 replicates of B. juncea and 5 replic~tes o/B. carinata. (mg Kg-1dry weight) 5.91 14.76 1.62 7.51 14.91 0.07

B. carinata (nc) B. juncea (nc) B. carinata B. juncea

4,43 6,23

~4,14 1,08

6,70 5,82

Significative concentrations of Cd were not detected nc: no contaminated soil

It is well known that multiple interactions among t~e metals in the soil can affect the capacity of the

M. del Ro et al.

331

plant for uptaking, i.e. studies have shown that the prese e of Cd can increase the accumulation of Zn in some species7, and in addition Cu decreases Zn ptake in the soil5. This could explain the differences in Cu content between plants grown in no contaminated soil ITom a locality of the Guadiamar river afea and those grown in contaminated s il. The first one exhibited values of 14.91 mg Kg-l in the case of B. juncea and 14.76 mg Kg-l fo B. carinata which are higher than those found in plants grown in contaminated soils (6.70 and 5. 2. mg Kg-\ respectively). Both B. juncea and B. carinata accumulated the highe content of Pb and Cu in the leaves (pb 55.21%; Cu 34.53% and Pb 46.10%; Cu 39.79% in B. ju cea and B. carinata, respectively. Similar results were obtained by Tlustos8 for Cd, who investi ated the uptake of this metal by radish (Raphanus sativus L), found the highest concentraran o~Cd in the leaves ofthis species. The majar part of Zn was concentrated in the stems (~8.62% and 38.92% in B. juncea and B. carinata, respectively) (Table 3).

Table 3. Percentages ofPb, Zn and Cu in Brassica carin'pta and Brassica juncea leaves, stem, pod, seeds and root. Results ate means of 25 replicates of B. juncea and 126 replicates o/B. carinata. Species pars (%) Cu Pb Zn B. carinata 46,10 31,21 39,79 leaf stem 32,47 38,92 28,13 3,82 2,83 3,39 pod and seed root 18,60 26,48 28,26 55,21 26,91 B.juncea 34,53 leaf stem 26,46 22,91 48,62 17,03 12,71 21,91 pod seed 22,55 3,22 11,64 root 3,57 3,21 4,78 In B. juncea the levels of Pb and Cu were in the arder leaves>stem>pods>seeds>roots. exception was Zn which had concentrations in stem>leaves>pods>seeds>roots The only

due to the higher

biomass of the stem. B carinata showed the same relation but pods and seeds had the lowest levels for al! the metals due to the sowing conditions described previously. These results show that in spite of the unfavourable agronomic conditions since the sowing until the harvest, these species concentrated Pb, Zn and Cu contents much higher than the soluble metal levels present in the soil. To improve the agronomic conditions in the sowing and growing times, will bring an increase ofthe phytoextraction capacity by these species

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M. del Rio et al.

ACKNO\YLEDGEl\'IENTS The authors thank Consejo Superior de Investigaciones Cientficas (CSIC) and Consejera de Medio Ambiente (Junta de Andaluca) for supporting this research, and Gloria Femndez Martnez (Instituto de Agricultura Sostenible, CSIC, Crdoba), for her help in performing the analyses of soil and plants.

REFERENCES
1-Kumar, P.B.A. N. 1995. Phytoextraction: The use of plants to remove heavy metals from soils. Environ. Sci. Techn. 29 (5): 1232-1238. 2- Brooks, RR., Lee, J., Reeves, R.D., Jaffre, T. 1977. Detection of nickeliferous rocks by analysis ofherbarium specimens of indicator plants. J. Geochem. Explor. 7: 49-57. 3- Baker, AJ.M., Reeves, R.D., Rajar, A S.M. 1994. Reavy metal accumulation and tolerance in British population of the metallophyte Thlaspi caerulescens. J. & Presl. (Brassicaceae). New Phytol. 127: 61-68. 4-Kumar, P.B.A N., Dushenkov, V. and Ensley, B.D., Raskin, 1. 1995a. The use of crop Brassicas in phytoeXtraction: a subset of phytoremediation to remo ve toxic metals from soils. In: Ninth Intemational Rapeseed Congress. Cambridge, UK. 4 to 7 July. Vol. 3. pp: 753-756. 5-Ebbs, S.D. and Kochian, Lv. 1997. Toxicity ofzinc and correr to Brassica species: implications for phytoremediation. J. Environ. Qual. 26: 776-781 6-Salt, D.E., Blaylock, M., Kumar, N.P.B.A., Dushenkov, V., Ensley, B.D., Chet, 1. 1995 Phytoremediation: A novel strategy for the removal of toxic metals from the enviroment using plants. Biotechnology. 13: 468-474. 7-Tumer, ~f.A. 1973. Effect of cadmium treatment on cadmium and zinc uptake by selected vegetable species. J. Environ. Qual. 2: 118-119. 8-Tlustos, P., Pavlkov, D., Balk, J., Szkov, J., Rane, A., Balkov, M. 1998. The aeeumulation ofarsenie and cadmium in plants and their distribution. Rostlinn Vyroba. 44 (lO): 463-469. Received for publieation: Accepted lar publication: November 08, 1999 May 08,2000