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Seasonal and annual variations of metal uptake, bioaccumulation, and toxicity in Trifolium repens and Lolium perenne growing in a heavy metal-contaminated field

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Background, aim, and scope

The reclamation of nonferrous metal-polluted soil by phytoremediation requires an overall and permanent plant cover. To select the most suitable plant species, it is necessary to study metal effects on plants over the time, thereby checking that metals remain stored in root systems and not transferred to aerial parts. In this purpose, the seasonal and annual variations of metal bioaccumulation, transfer, and phytotoxicity in Trifolium repens and Lolium perenne grown in a Cd-, Pb-, and Zn-contaminated soil were also studied.

Materials and methods

The experimental site was located near a closed smelter. In spring 2004, two areas were sown with T. repens and L. perenne, respectively. Thereafter, the samplings of plant roots and shoots and surrounding soils were realized in autumn 2004 and spring and autumn 2005. The soil agronomic characteristics, the Cd, Pb, and Zn concentrations in the surrounded soils and plant organs, as well as the oxidative alterations (superoxide dismutase [SOD], malondialdehyde [MDA], and 8-hydroxy-2′-deoxyguanosine [8-OHdG]) in plant organs were carried out.


Whatever the sampling period, metal concentrations in soils and plants were higher than background values. Contrary to the soils, the fluctuations of metal concentrations were observed in plant organs over the time. Bioaccumulation and transfer factors confirmed that metals were preferentially accumulated in the roots as follows: Cd>Zn>Pb, and their transfer to shoots was limited. Foliar metal deposition was also observed. The results showed that there were seasonal and annual variations of metal accumulation in the two studied plant species. These variations differed according to the organs and followed nearly the same pattern for the two species. Oxidative alterations were observed in plant organs with regard to SOD antioxidant activities, MDA, and 8-OHdG concentrations. These alterations vary according to the temporal variations of metal concentrations.


Metal concentrations in surrounded soils and plant organs showed the effective contamination by industrial dust emissions. Metals absorbed by plants were mainly stored in the roots. With regard to this storage, the plants seemed to limit the metal transfer to their aerial parts over the time, thereby indicating their availability for metal phytostabilization. Aerial deposition was another source of plant exposure to nonferrous metals. Despite the occurrence of metal-induced oxidative alterations in plant organs, both plant species seemed to tolerate a high metal concentration in soils.


Taken together, these results indicated that T. repens and L. perenne were able to form a plant cover on highly Cd-, Pb-, and Zn-polluted soils, to limit the metal transfer to their aerial parts and were relatively metal-tolerant. All these characteristics made them suitable for phytostabilization on metal-contaminated soils. These findings also highlighted the necessity to take into account seasonal and annual variations for a future phytomanagement.

Recommendations and perspectives

In this work, the behavior of plant species grown in metal-polluted soil has been studied during 2 years. Obviously, this time is too short to ensure that metals remain accumulated in the root system and few are transferred in aerial parts over the time. It is why regular monitoring should be achieved during more than a decade after the settlement of the plant cover. This work will be completed by the study of the T. repens and L. perenne effects on mobility of metals in order to evaluate the quantities of pollutants which could be absorbed by the biota and transferred to groundwater. Bioaccessibility tests could be also realized on polluted soils in order to evaluate the phytostabilization impacts on the exposition risks for humans.

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  1. Alfani A, Maisto G, Iovieno P, Rutigliano FA, Bartoli G (1996) Leaf contamination by atmospheric pollutants as assessed by elemental analysis of tissue, leaf surface deposit and soil. J Plant Physiol 148:243–248

  2. An YJ (2004) Soil ecotoxicity assessment using cadmium sensitive plants. Environ Sci Technol 127:21–26

  3. Bidar G, Garçon G, Pruvot C, Dewaele D, Cazier F, Douay F, Shirali P (2007) Behavior of Trifolium repens and Lolium perenne growing in a heavy metal contaminated field: plant metal concentration and phytotoxicity. Environ Pollut 147:546–553

  4. Brekken A, Steinnes E (2004) Seasonal concentrations of cadmium and zinc in native pasture plants: consequences for grazing animals. Sci Total Environ 326:181–195

  5. Chaney RL, Malik M, Li YM, Brown SL, Brewer EP, Angle JS, Baker AJM (1997) Phytoremediation of soil metals. Curr Opin Biotechnol 8:279–284

  6. Chaoui A, Mazhoudi S, Ghorbal MH, El Ferjani E (1997) Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseolus vulgaris L.). Plant Sci 127:139–147

  7. Cheng S (2003) Heavy metals in plants and phytoremediation. Environ Sci Pollut Res 10:335–340

  8. Dahmani-Muller H, van Oort F, Gélie B, Balabane M (2000) Strategies of heavy metal uptake by three plant species growing near a metal smelter. Environ Pollut 109:231–238

  9. Davranche M, Bollinger JC (2001) A desorption-dissolution model for metal release from polluted soil under reductive conditions. J Environ Qual 30:1581–1586

  10. Deram A, Denayer FO, Petit D, Van Haluwyn C (2006) Seasonal variations of cadmium and zinc in Arrhenatherum elatius, a perennial grass species from highly contaminated soils. Environ Pollut 140:62–70

  11. Dietl C, Wäber M, Peichl L, Vierle O (1996) Monitoring of airborne metals in grass and depositions. Chemosphere 33:2101–2111

  12. Dixit V, Pandey V, Shyam R (2001) Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). J Exp Bot 52:1101–1109

  13. Djingova R, Kuleff I (1994) On the sampling of vascular plants for monitoring of heavy metal pollution. In: B. Markert (Ed.) Environmental sampling for trace analysis. VCH, Weinheim, pp 395–414

  14. Douay F, Roussel H, Pruvot C, Waterlot C (2008) Impact of the smelter closedown on metal contents of wheat cultivated in the neighbourhood. Environ Sci Pollut Res 15:162–169

  15. DRIRE (2003) L’industrie au regard de l’environnement en 2002. Douai. Direction Régionale de l’Industrie de la Recherche et de l’Environnement, 308 pp

  16. Duman F, Cicek M, Sezen G (2007) Seasonal changes of metal accumulation and distribution in common club rush (Schoenoplectus lacustris) and common reed (Phragmites australis). Ecotoxicology 16:457–463

  17. Erhola M, Toyokuni S, Okada K, Tanaka T, Hiai H, Ochi H, Uchida K, Osawa T, Nieminen MM, Alho H, Kellokumpu-Lehtinen P (1997) Biomarker evidence of DNA oxidation in lung cancer patients: association of urinary 8-hydroxy-2′-deoxyguanosine excretion with radiotherapy, chemotherapy, and response to treatment. FEBS Lett 409:287–291

  18. François M, Dubourguier HC, Li D, Douay F (2004) Prediction of heavy metal solubility in agricultural topsoils around two smelters by the physico-chemical parameters of the soils. Aquat Sci 66:78–85

  19. Gallego SM, Benavídes MP, Tomaro ML (1996) Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Sci 121:151–159

  20. Garçon G, Garry S, Gosset P, Zerimech F, Martin A, Hannothiaux M, Shirali P (2001) Benzo(a)pyrene-coated onto Fe2O3 particles-induced lung tissue injury: role of free radicals. Cancer Lett 167:7–15

  21. Garçon G, Dagher Z, Zerimech F, Ledoux F, Courcot D, Aboukais A, Puskaric E, Shirali P (2006) Dunkerque city air pollution particulate matter-induced cytotoxicity, oxidative stress and inflammation in human epithelial lung cells (L132) in culture. Toxicol In Vitro 20:519–528

  22. Grant CA, Buckley WC, Bailey LD, Selles F (1997) Cadmium accumulation in crops. Can J Plant Sci 78:1–17

  23. Hagemeyer J, Schäfer H (1995) Seasonal variations in concentrations and radial distribution patterns of Cd, Pb and Zn in stem wood of beech trees (Fagus sylvatica L). Sci Total Environ 166:77–87

  24. Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604–611

  25. Kabata-Pendias A, Pendias H (2001) Trace elements in soils and plants. CRC, Boca Raton, FL, p 413

  26. Kim ND, Fergusson JE (1994) Seasonal variations in the concentrations of cadmium, copper, lead and zinc in leaves of the horse chesnut (Aesculus hippocastanum L). Environ Pollut 86:89–97

  27. Kohler M, Gleisberg B, Niese S (2000) Investigation of the soil–plant transfer of primordial radionuclides in tomatoes by low-level g-ray spectrometry. Appl Radiat Isotopes 53:203–208

  28. Kuchino Y, Mori F, Kasai H, Inoue H, Iwai S, Miura K, Ohtsuka E, Nishimura S (1987) Misreading of DNA templates containing 8-hydroxydeoxy-guanosine at the modified base and adjacent residues. Nature 327:77–79

  29. Lima AIG, Pereira SIA, Figueira EMAP, Caldeira GCN, Caldeira HDQM (2006) Cadmium detoxification in roots of Pisum sativum seedlings: relationship between toxicity levels, thiol pool alterations and growth. Environ Exp Bot 55:149–162

  30. Matthews H, Thornton J (1982) Seasonal and species variation in the content of cadmium and associated metals in pasture plants at Shipham. Plant Soil 66:181–193

  31. McGrath D (1996) Application of single and sequential extraction procedures to polluted and unpolluted soils. Sci Total Environ 178:37–44

  32. Milone MT, Sgherri C, Clijsters H, NavariIzzo F (2003) Antioxidative responses of wheat treated with realistic concentration of cadmium. Environ Exp Bot 50:265–276

  33. Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410

  34. Moller P, Wallin H (1998) Adduct formation, mutagenesis and nucleotide excision repair of DNA damage produced by reactive oxygen species and lipid peroxidation product. Mutat Res 410:271–290

  35. Mulligan CN, Yong RN, Gibbs BF (2001) Remediation technologies for metal-contaminated soils and groundwater—an evaluation. Eng Geol 60:193–207

  36. Munné-Bosch S (2005) The role of a-tocopherol in plant stress tolerance. J Plant Physiol 162:743–748

  37. Pruvot C, Douay F, Fourrier H, Waterlot C (2006) Heavy metals in soil, crops, and grass as a source of human exposure in the former mining areas. J Soils Sediments 6:215–220

  38. Reddy BK, Kumar GNM, Jyothsnakumari G, Thimmanaik S, Sudhakar C (2005) Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.). Chemosphere 60:97–104

  39. Remon E, Bouchardon JB, Cornier B, Guy B, Leclerc JC, Faure O (2005) Soil characteristics, heavy metal availability and vegetation recovery at a former metallurgical landfill, implications in risk assessment and site restoration. Environ Pollut 137:316–323

  40. Rieuwerts J, Farago M (1996) Heavy metal pollution in the vicinity of a secondary lead smelter in the Czech Republic. Appl Geochem 11:17–23

  41. Ruley AT, Sharma NC, Sahi SV (2004) Antioxidant defense in lead accumulating plant, Sesbania drummondii. Plant Physiol Biochem 42:899–906

  42. Salt DE, Smith RD, Raskin I (1998) Phytoremediation. Annu Rev Plant Physiol Plant Mol Biol 40:643–668

  43. Schröder P, Fischer C, Debus R, Wenzel A (2003) Reaction of detoxification mechanisms in suspension cultured spruce cells (Picea abies L. Karst.) to heavy metals in pure mixture and in soil eluates. Environ Sci Pollut Res 10:225–234

  44. Schützendübel A, Polle A (2002) Plant responses to abiotic stresses, heavy metal-induced oxidative stress and protection by mycorrhization. J Exp Bot 53:1351–1365

  45. Souza JF, Rauser WE (2003) Maize and radish sequester excess cadmium and zinc in different ways. Plant Sci 165:1009–1022

  46. Sterckeman T, Douay F, Proix N, Fourrier H (2000) Vertical distribution of Cd, Pb and Zn in soils near smelters in the North of France. Environ Pollut 107:377–389

  47. Sterckeman T, Douay F, Proix N, Fourrier H, Perdrix E (2002a) Assessment of the contamination of cultivated soils by eighteen trace elements around smelters in the north of France. Water Air Soil Pollut 135:173–194

  48. Sterckeman T, Douay F, Fourrier H, Proix N (2002b) Référentiel pédo-géochimique du Nord-Pas de Calais. Technical report, Conseil Régional Nord-Pas de Calais-Ministère de l’Aménagement du Territoire et de l’Environnement, 128 pp

  49. Toyokuni S, Tanaka T, Hatturi Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, Osawa T (1997) Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N451—its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest 76:365–374

  50. Vangronsveld J, Sterckxb J, Van Asschec F, Clijstersa H (1995) Rehabilitation studies on an old non-ferrous waste dumping ground, effects of revegetation and metal immobilization by beringite. J Geochem Explor 52:221–229

  51. Verma S, Dubey RS (2003) Lead toxicity induces lipid peroxidation and alters the activities of antioxidant enzymes in growing rice plants. Plant Sci 164:645–655

  52. Wong MH (2003) Ecological restoration of mine degraded soils, with emphasis on metal contaminated soils. Chemosphere 50:775–780

  53. Wu F, Zhang G, Dominy P (2003) Four barley genotypes respond differently to cadmium: lipid peroxidation and activities of antioxidant capacity. Environ Exp Bot 50:67–78

  54. Ye ZH, Yang ZY, Chan GYS, Wong MH (2001) Growth response of Sesbania rostrata and S cannabina to sludge-amended lead/zinc mine tailings—a greenhouse study. Environ Int 26:449–455

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We are grateful to the Etablissement Public Foncier Nord-Pas de Calais and the industrial partners (EDF, Surschiste, APINOR) for the settlement of the experimental site and to ADEME for its financial contribution.

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Correspondence to Francis Douay.

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Bidar, G., Pruvot, C., Garçon, G. et al. Seasonal and annual variations of metal uptake, bioaccumulation, and toxicity in Trifolium repens and Lolium perenne growing in a heavy metal-contaminated field. Environ Sci Pollut Res 16, 42–53 (2009). https://doi.org/10.1007/s11356-008-0021-4

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  • Bioaccumulation factors
  • L. perenne
  • Nonferrous metal-contaminated soil
  • Oxidative stress
  • Phytostabilization
  • Seasonal and annual variations
  • T. repens
  • Transfer factors