TiO2 nanoparticles may alleviate cadmium toxicity in co-treatment experiments on the model hydrophyte Azolla filiculoides

  • Carmelina SpanòEmail author
  • Stefania Bottega
  • Carlo Sorce
  • Giacomo Bartoli
  • Monica Ruffini Castiglione
Research Article


The hydrophyte Azolla filiculoides can be a useful model to assess if TiO2 NPs may in some way alleviate the Cd injuries and improve the ability of the plant to cope with this metal. With this mechanistic hypothesis, after a pre-treatment with TiO2 NPs, A. filiculoides plants were transferred to cadmium-contaminated water with or without TiO2 nanoparticles. After 5 days of treatment, cadmium uptake, morpho-anatomical, and physiological aspects were studied in plants. The continuous presence of TiO2 nanoparticles, though not increasing the uptake of cadmium in comparison with a priming treatment, induced a higher translocation of this heavy metal to the aerial portion. Despite the translocation factor was always well below 1, cadmium contents in the fronds, generally greater than 100 ppm, ranked A. filiculoides as a good cadmium accumulator. Higher cadmium contents in leaves did not induce damages to the photosynthetic machinery, probably thanks to a compartmentalization strategy aimed at confining most of this pollutant to less metabolically active peripheral cells. The permanence of NPs in growth medium ensured a better efficiency of the antioxidant apparatus (proline and glutathione peroxidase and catalase activities) and induced a decrease in H2O2 content, but did not suppress TBARS level.


Azolla filiculoides Cadmium Histochemistry Oxidative stress Photosynthetic efficiency TiO2 nanoparticles 


Funding information

This work was supported by local funding of the University of Pisa (ex 60 %).

Supplementary material

11356_2019_6148_MOESM1_ESM.jpg (4 mb)
Fig. 1S Photo of experimental setup and plants (JPG 4145 kb)
11356_2019_6148_Fig5_ESM.png (6.8 mb)
Fig. 2S

In situ Cd localization in A. filiculoides roots and mature leaves after treatment with dithizone. Root images correspond to a portion about 2 mm far from the apex except for g and for insert in e (about 5 mm from the apex). a) Detail of the root and root hairs of C plants. b) ventral side of the dorsal lobe of a C leaf: Mm: membranous margin of the foliar lobe; Cr: thick photosynthetic central region; Cv: pore of the leaf cavity. c) Detail of the root and root hairs of CNPs plants. d) feature of Cr and Mm of the leaf lobe in CNPs plants. e-f) Cd5 treatment: root and leaf representative images with brown/reddish Cd precipitates (arrows). g-h) Cd10 treatment: root and leaf representative images with brown/reddish Cd precipitates (arrows). i-j) Cd5+NPs treatment: root and leaf representative images with brown/reddish Cd precipitates (arrows); Cd positivity is also detectable in dorsal epidermis and papillae (Pp). k-l) Cd10+NPs treatment: root and leaf representative images with brown/reddish Cd precipitates (arrows); Cd positivity is also detectable in dorsal epidermis and papillae (Pp). C=deionized water; CNPs=TiO2 NPs; Cd5=5 mg L−1 CdCl2; Cd10=10 mg L−1 CdCl2; Cd5+NPs = 5 mg L−1 CdCl2 plus TiO2 NPs; Cd10+NPs = 10 mg L−1 CdCl2 plus TiO2NPs. (PNG 7013 kb)

11356_2019_6148_MOESM2_ESM.tif (35 mb)
High Resolution Image (TIF 35789 kb)


  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126CrossRefGoogle Scholar
  2. Arezki O, Boxus P, Kevers C, Gaspar T (2001) Changes in peroxidase activity: and level of phenolic compounds during light-induced plantlet regeneration from Eucalyptus camaldulensis Dhen. nodes in vitro. Plant Growth Regul 33:215–219CrossRefGoogle Scholar
  3. ATSDR (2012) Toxicological profile for cadmium.
  4. Augstein F, Carlsbecker A (2018) Getting to the roots: a developmental genetic view of root anatomy and function from Arabidopsis to Lycophytes. Front Plant Sci 9:1410CrossRefGoogle Scholar
  5. Bahmani R, Kim DG, Kim JA, Hwang S (2016) The density and length of root hairs are enhanced in response to cadmium and arsenic by modulating gene expressions involved in fate determination and morphogenesis of root hairs in Arabidopsis. Front Plant Sci 7:1763CrossRefGoogle Scholar
  6. Balestri M, Bottega S, Spanò C (2014a) Response of Pteris vittata to different cadmium treatments. Acta Physiol Plant 36:767–775CrossRefGoogle Scholar
  7. Balestri M, Ceccarini A, Forino LMC, Zelko I, Martinka M, Lux A, Ruffini Castiglione M (2014b) Cadmium uptake, localization and stress-induced morphogenic response in the fern Pteris vittata. Planta 239:1055–1064CrossRefGoogle Scholar
  8. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  9. Chang ML, Chen NY, Liao LJ, Cho CL, Liu ZH (2012) Effect of cadmium on peroxidase isozyme activity in roots of two Oryza sativa cultivars. Bot Stud 53:31–44Google Scholar
  10. Ciobanu C, Şlencu BG, Cuciureanu R (2013) FAAS determination of cadmium and lead content in foodstuffs from north-eastern Romanian market. Studia Universitatis “Vasile Goldiş”, Seria Ştiinţele Vieţii 23:33–38Google Scholar
  11. Cuypers A, Smeets K, Ruytinx J, Opdenakker K, Keunen E, Remans T, Horemans N, Vanhoudt N, Sanden SV, Belleghem FV, Guisez Y, Colpaertm J, Vangronsveld J (2011) The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings. J Plant Physiol 168:309–316CrossRefGoogle Scholar
  12. Dai LP, Xiong ZT, Huang Y, Li MJ (2006) Cadmium-induced changes in pigments, total phenolics, and phenylalanine ammonia-lyase activity in fronds of Azolla imbricata. Environ Toxicol 21:505–512CrossRefGoogle Scholar
  13. Daudi A, Cheng Z, O’Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP (2012) The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant Cell 24:275–287CrossRefGoogle Scholar
  14. Deng R, Lin D, Zhu L, Majumdar S, White JC, Gardea-Torresdey JL, Xing B (2017) Nanoparticle interactions with co-existing contaminants: joint toxicity, bioaccumulation and risk. Nanotoxicology 11:591–612CrossRefGoogle Scholar
  15. Forino LMC, Ruffini Castiglione M, Bartoli G, Balestri M, Andreucci A, Tagliasacchi AM (2012) Arsenic-induced morphogenic response in roots of arsenic hyperaccumulator fern Pteris vittata. J Hazard Mater 235–236:271–278CrossRefGoogle Scholar
  16. Gallego SM, Pena LB, Barcia RA, Azpilicueta CE, Iannone MF, Rosales EP, Zawoznik MS, Groppa MD, Benavides MP (2012) Unravelling cadmium toxicity and tolerance in plants: insight into regulatory mechanisms. Environ Exp Bot 83:33–46CrossRefGoogle Scholar
  17. Genty B, Briantais JM, Baker NR (1989) The relationship between quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92CrossRefGoogle Scholar
  18. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930CrossRefGoogle Scholar
  19. Giordani T, Fabrizi A, Guidi L, Natali L, Giunti G, Ravasi F, Cavallini A, Pardossi A (2012) Response of tomato plants exposed to treatment with nanoparticles. Int J Environ Qual 8:27–38Google Scholar
  20. Giorgetti L, Spanò C, Muccifora S, Bellani L, Tassi E, Bottega S, Di Gregorio S, Siracusa G, Sanità di Toppi L, Ruffini Castiglione M (2019) An integrated approach to highlight biological responses of Pisum sativum root to nano-TiO2 exposure in a biosolid-amended agricultural soil. Sci Total Environ 650:2705–2716CrossRefGoogle Scholar
  21. Hassanzadeh M, Ebadi A, Panahyan-e-Kivi M, Eshghi AG, Jamaati-e-Somarin S, Saeidi M, Zabihi-e-Mahmoodabad R (2009) Evaluation of drought stress on relative water content and chlorophyll content of sesame (Sesamum indicum L.) genotypes at early flowering stage. Res J Environ Sci 3:345–350CrossRefGoogle Scholar
  22. Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A (2012) Role of proline under changing environments. A review. Plant Signal Behav 7:1456–1466CrossRefGoogle Scholar
  23. Irfan M, Ahmad A, Hayat S (2014) Effect of cadmium on the growth and antioxidant enzymes in two varieties of Brassica juncea. Saudi J Biol Sci 21:125–131CrossRefGoogle Scholar
  24. Jana S, Choudhuri MA (1982) Glycolate metabolism of three submerged aquatic angiosperms during aging. Aquat Bot 12:345–354CrossRefGoogle Scholar
  25. Ji Y, Zhou Y, Ma C, Feng Y, Hao Y, Rui Y, Wu W, Gui X, Le VN, Han Y, Wang Y, Xing B, Liu L, Cao W (2017) Jointed toxicity of TiO2 NPs and Cd to rice seedlings: NPs alleviated Cd toxicity and Cd promoted NPs uptake. Plant Physiol Biochem 110:82–93CrossRefGoogle Scholar
  26. Kapoor D, Rattan A, Bhardwaj R, Kaur S, Gupta A, Manoj (2016) Antioxidative defense responses and activation of phenolic compounds in Brassica juncea plants exposed to cadmium stress. Int J Green Pharm 10:228-234Google Scholar
  27. Kováčik J (2013) Hyperaccumulation of cadmium in Matricaria chamomilla: a never-ending story? Acta Physiol Plant 35:1721–1725CrossRefGoogle Scholar
  28. Lei Z, Mingyu S, Xiao W, Chao L, Chunxiang Q, Liang C, Hao H, Xiaoqing L, Fashui H (2008) Antioxidant stress is promoted by nano-anatase in spinach chloroplasts under UV-B radiation. Biol Trace Elem Res 121:69–79CrossRefGoogle Scholar
  29. Leng BY, Jia WJ, Yan X, Yuan F, Dong XX, Wang BS (2018) Cadmium stress in halophyte Thellungiella halophila: consequences on growth, cadmium accumulation, reactive oxygen species and antioxidative systems. IOP Conf Ser: Earth Environ Sci 153:062002CrossRefGoogle Scholar
  30. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382CrossRefGoogle Scholar
  31. Lopez-Luna J, Silva-Silva MJ, Martinez-Vargas S, Mijangos-Ricardez OF, Gonzalez-Chavez MC, Solis-Dominguez FA, Cuevas-Diaz MC (2016) Magnetite nanoparticle (NP) uptake by wheat plants and its effect on cadmium and chromium toxicological behavior. Sci Total Environ 565:941–950CrossRefGoogle Scholar
  32. Lyu S, Wei X, Chen J, Wang C, Wang X, Pan D (2017) Titanium as a beneficial element for crop production. Front Plant Sci 8:597CrossRefGoogle Scholar
  33. Manesh RR, Grassi G, Bergami E, Marques-Santos LF, Faleri C, Liberatori G, Corsi I (2018) Co-exposure to titanium dioxide nanoparticles does not affect cadmium toxicity in radish seeds (Raphanus sativus). Ecotoxicol Environ Saf 148:359–366CrossRefGoogle Scholar
  34. Naghipour D, Ashrafi SD, Gholamzadeh M, Taghavi K, Naimi-Joubani M (2018) Phytoremediation of heavy metals (Ni, Cd, Pb) by Azolla filiculoides from aqueous solution: a dataset. Data in Brief 21:1409–1414CrossRefGoogle Scholar
  35. Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880Google Scholar
  36. Natarajan A, Vijayarengan P, Vijayaragavan M (2018) Influence of cadmium on growth and biochemical contents of tomato plants. J Plant Stress Physiol 4:4-6Google Scholar
  37. Navari-Izzo F, Meneguzzo S, Loggini B, Vazzana C, Sgherri CLM (1997) The role of the glutathione system during dehydration of Boea hygroscopica. Physiol Plant 99:23–30CrossRefGoogle Scholar
  38. Okupnik A, Pflugmacher S (2016) Oxidative stress response of the aquatic macrophyte Hydrilla verticillata exposed to TiO2 nanoparticles. Environ Toxicol Chem 35:2859–2866CrossRefGoogle Scholar
  39. Pielichowska M, Wierzbicka M (2004) Uptake and localization of cadmium by Biscutella laevigata, a cadmium hyperaccumulator. Acta Biol Cracov Ser Bot 46:57–63Google Scholar
  40. Piñeros MA, Shaff JE, Kochian V (1998) Development, characterization, and application of a cadmium-selective microelectrode for the measurement of cadmium fluxes in roots of Thlaspi species and wheat. Plant Physiol 116:1393–1401CrossRefGoogle Scholar
  41. Praetorius A, Scheringer M, Hungerbühler K (2012) Development of environmental fate models for engineered nanoparticles- a case study of TiO2 nanoparticles in the Rhine river. Environ Sci Technol 46:6705–6713CrossRefGoogle Scholar
  42. Prasad SM, Singh A (2011) Metabolic responses of Azolla pinnata to cadmium stress: photosynthesis, antioxidative system and phytoremediation. Chem Ecol 27:543–555CrossRefGoogle Scholar
  43. Rao MV, Beverley AH, Ormrod DP (1995) Amelioration of ozone-induced oxidative damage in wheat plants grown under high carbon dioxide. Role of antioxidant enzymes. Plant Physiol 109:421–432CrossRefGoogle Scholar
  44. Rossi L, Sharifan H, Zhang W, Schwab AP, Ma X (2018) Mutual effects and in planta accumulation of co-existing cerium oxide nanoparticles and cadmium in hydroponically grown soybean (Glycine max (L.) Merr.). Environ Sci Nano 5:150–157Google Scholar
  45. Ruffini Castiglione M, Giorgetti L, Cremonini R, Bottega S, Spanò C (2014) Impact of TiO2 nanoparticles on Vicia narbonensis L.: potential toxicity effects. Protoplasma 251:1471–1479CrossRefGoogle Scholar
  46. Ruffini Castiglione M, Giorgetti L, Bellani L, Muccifora S, Bottega S, Spanò C (2016) Root responses to different types of TiO2 nanoparticles and bulk counterpart in plant model system Vicia faba L. Environ Exp Bot 130:11–21CrossRefGoogle Scholar
  47. Servin AD, Morales MI, Castillo-Michel H, Hernandez-Viezcas JA, Munoz B, Zao L, Nunez JE, Peralta-Videa JR, Gardea-Torresdey JL (2013) Synchrotron verification of TiO2 accumulation in cucumber fruit: a possible pathway of TiO2 nanoparticle transfer from soil into the food chain. Environ Sci Technol 47:11592–11598CrossRefGoogle Scholar
  48. Sood A, Uniyal PL, Prasanna R, Ahluwalia AS (2012) Phytoremediation potential of aquatic macrophyte, Azolla. Ambio 41:122–137CrossRefGoogle Scholar
  49. Spanò C, Bottega S (2016) Durum wheat seedlings in saline conditions: salt spray versus root-zone salinity. Estuar Coast Shelf Sci 169:173–181CrossRefGoogle Scholar
  50. Spanò C, Bruno M, Bottega S (2013) Calystegia soldanella: dune versus laboratory plants to highlight key adaptive physiological traits. Acta Physiol Plant 35:1329–1336CrossRefGoogle Scholar
  51. Spanò C, Bottega S, Ruffini Castiglione M, Pedranzani HE (2017) Antioxidant response to cold stress in two oil plants of the genus Jatropha. Plant Soil Environ 63:271–276CrossRefGoogle Scholar
  52. Tan C-y, Shan X-q, Xu G-z, Lin YM, Chen Z-l (2011) Phytoaccumulation of cadmium through Azolla from aqueous solution. Ecol Eng 37:1942–1946CrossRefGoogle Scholar
  53. Valderrama A, Carvajal DE, Peñailillo P, Tapia J (2016) Accumulation capacity of cadmium and copper and their effects on photosynthetic performance in Azolla filiculoides Lam. under induced rhizofiltration. Gayana Bot 73:283–291CrossRefGoogle Scholar
  54. Wang Z, Zhang Y, Huang Z, Huang L (2008) Antioxidative response of metal-accumulator and non-accumulator plants under cadmium stress. Plant Soil 310:137–149CrossRefGoogle Scholar
  55. Wang Q, Wang L, Han R, Yang L, Zhou Q, Huang X (2015) Effects of bisphenol A on antioxidant system in soybean seedling roots. Environ Toxicol Chem 34:1127–1133CrossRefGoogle Scholar
  56. Weir A, Westerhoff P, Fabricius L, Hristovski K, von Goetz N (2012) Titanium dioxide nanoparticles in food and personal care products. Environ Sci Technol 46:2242–2250CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Carmelina Spanò
    • 1
    Email author
  • Stefania Bottega
    • 1
  • Carlo Sorce
    • 1
  • Giacomo Bartoli
    • 1
  • Monica Ruffini Castiglione
    • 1
  1. 1.Department of BiologyUniversity of PisaPisaItaly

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