Update on the Assessment of Chronic Phytotoxicity Using Fern Spore Biomarkers

  • Helena García-Cortés
  • Myriam Catalá
  • José Luis Rodríguez-Gil


The use of an adequate range of taxa in ecotoxicological studies is a key point for the achievement of ecologically relevant results. Higher plants are an essential part of a healthy and balanced ecosystem, and new plant models are essential in the evaluation of potential impacts of pollutants. With more than 10,000 living species, ferns are the second largest group of vascular plants. Fern spores and spore-developed gametophytes have long been recognized as useful models in many important areas of plant research. Fern spores are single meiotic cells that develop into gametophytes which are miniature mature higher plants. The use of microtubes and microplates is imposed by the natural model. Chronic toxicity testing involves longer periods of exposure to toxicants (>48 h) and assesses the ability of a substance to disrupt a significant portion of an organism’s life stage. DNA content in developing gametophytes can be used as a biomarker of the disturbance that the toxicant provokes in gametophyte growth and development. Chlorophyll autofluorescence can also be used as a biomarker of the physiological state. Both biomarkers can easily be measured using 96 multiwell plates and plate readers. The combined use of these biomarkers in chronic toxicity tests using developing gametophytes of the riparian Polystichum setiferum is yielding very satisfactory results and is a promising new model for ecotoxicology. This bioassay has been successfully used in environmental toxicology/ecotoxicology, assessment of environmental technology and environmental monitoring studies.


Fern spores Chronic/subchronic phytotoxicity DNA Chlorophyll a Environmental monitoring Environmental technology 


  1. 15 U.S.C. §2601, et seq (1976) The toxic substances control actGoogle Scholar
  2. Agati G (1998) Response of the in vivo chlorophyll fluorescence spectrum to environmental factors and laser excitation wavelength. Pure Appl Opt 7:797–807CrossRefGoogle Scholar
  3. Banks JA (1999) Gametophyte developement in ferns. Annu Rev Plant Phys 50:163–186CrossRefGoogle Scholar
  4. Benenati FE (1990) Keynote address: plants - Kesystone to risk assessment. In: Wang W-c, Gorsuch JW, Lower WR (eds) Plants for toxicity assessment. ASTM, Philadelphia, pp 5–14CrossRefGoogle Scholar
  5. Boutin C, Aya KL, Carpenter D, Thomas PJ, Rowland O (2012) Phytotoxicity testing for herbicide regulation: shortcomings in relation to biodiversity and ecosystem services in agrarian systems. Sci Total Environ 415:79–92CrossRefPubMedGoogle Scholar
  6. Calabrese EJ (2008) Hormesis: why it is important to toxicology and toxicologists. Environ Toxicol Chem 27:1451–1474CrossRefPubMedGoogle Scholar
  7. Catala M, Esteban M, Rodríguez-Gil JL, Quintanilla LG (2009) Development of a naturally miniaturised testing method based on the mitochondrial activity of fern spores: a new higher plant bioassay. Chemosphere 77:983–988CrossRefPubMedGoogle Scholar
  8. Catalá M, Domínguez-Morueco N, Migens A, Molina R, Martínez F, Valcárcel Y, Mastroianni N, López de Alda M, Barceló D, Segura Y (2015) Elimination of drugs of abuse and their toxicity from natural waters by photo-Fenton treatment. Sci Total Environ 520:198–205CrossRefPubMedGoogle Scholar
  9. Chung KW, Fulton MH, Scott GI (2007) Use of juvenile clam Mercenaria Mercenaria, as a sensitive indicator of aqueus and sediment toxicity. Ecotoxicol Environ Saf 67:333–340CrossRefPubMedGoogle Scholar
  10. Czerniawska-Kusza I, Ciesielczuk T, Kusza G, Cichon A (2006) Comparison of the Phytotoxkit microbiotest and chemical variables for toxicity evaluation of sediments. Environ Toxicol 21:367–372CrossRefPubMedGoogle Scholar
  11. Daxhelet GA, Coene MM, Hoet PP, Cocito CG (1989) Spectrofluorometry of dyes with Dnas of Different Base composition and conformation. Anal Biochem 179:401–403CrossRefPubMedGoogle Scholar
  12. Doust JL, Schmidt M, Doust LL (1994) Biological assessment of aquatic pollution - a review, with emphasis on plants as biomonitors. Biol Rev 69:147–186CrossRefPubMedGoogle Scholar
  13. EFSA PPR Panel (EFSA Panel on Plant Protection Products and their Residues) (2014) Scientific opinion addressing the state of the science on risk assessment of plant protection products for non-target terrestrial plants. EFSA J 12(7):3800. 163 pp. CrossRefGoogle Scholar
  14. Esteban S, Fernández Rodríguez J, Díaz López G, Nuñez M, Valcárcel Y, Catalá M (2013) New microbioassays based on biomarkers are more sensitive to fluvial water micropollution than standard testing methods. Ecotoxicol Environ Saf 93:52–59CrossRefPubMedGoogle Scholar
  15. Esteban S, Llamas PM, García-Cortés H, Catalá M (2016) The endocrine disruptor nonylphenol induces sublethal toxicity in vascular plant development at environmental concentrations: a risk for riparian plants and irrigated crops? Environ Pollut 216:480–486CrossRefPubMedGoogle Scholar
  16. Evenari M (1949) Germination inhibitors. Bot Rev 15:153–194CrossRefGoogle Scholar
  17. Feito R, Valcárcel Y, Catalá M (2012) Biomarker assessment of toxicity with miniaturised bioassays: diclofenac as a case study. Ecotoxicology 21:289–296CrossRefPubMedGoogle Scholar
  18. Feito R, Valcárcel Y, Catalá M (2013) Preliminary data suggest that venlafaxine environmental concentrations could be toxic to plants. Chemosphere 90:2065–2069CrossRefPubMedGoogle Scholar
  19. Fent K, Weston AA, Caminada D (2006) Ecotoxicology of human pharmaceuticals. Aquat Toxicol 76:122–159CrossRefPubMedGoogle Scholar
  20. Ferrat L, Pergent-Martini C, Romeo M (2003) Assessment of the use of biomarkers in aquatic plants for the evaluation of environmental quality: application to seagrasses. Aquat Toxicol 65:187–204CrossRefPubMedGoogle Scholar
  21. Galbraith DW, Harkins KR, Jefferson RA (1988) Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts. Cytometry 9:75–83CrossRefPubMedGoogle Scholar
  22. García-Cambero JP, García-Cortés H, Valcárcel Y, Catalá M (2015) Environmental concentrations of the cocaine metabolite benzoylecgonine induced sublethal toxicity in the development of plants but not in a zebrafish embryo-larval model. J Hazard Mater 300:866–872CrossRefPubMedGoogle Scholar
  23. Khetan SK, Collins TJ (2007) Human pharmaceuticals in the aquatic environment: a challenge to green chemistry. Chem Rev 107:2319–2364CrossRefPubMedGoogle Scholar
  24. Krause GH, Weis E (1991) Chlorophyll fluorescence and photosynthesis - the basics. Annu Rev Plant Phys 42:313–349CrossRefGoogle Scholar
  25. Labarca C, Paigen K (1980) Simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344–352CrossRefPubMedGoogle Scholar
  26. Landis WG, Hughes JS, Lewis MA (1993) Environmental toxicology and risk assessment. ASTM, Philadelphia, 431 pCrossRefGoogle Scholar
  27. Lewis MA (1995) Use of fresh-water plants for Phytotoxicity testing - a review. Environ Pollut 87:319–336CrossRefPubMedGoogle Scholar
  28. Maiti S, Maiti P, Sinha SS, Mitra RK, Pal SK (2009) Molecular recognition of plant DNA: does it differ from conventional animal DNA? Int J Biol Macromol 44:133–137CrossRefPubMedGoogle Scholar
  29. Mocharla R, Mocharla H, Hodes ME (1987) A novel, sensitive fluorometric staining technique for the detection of DNA in RNA preparations. Nucleic Acids Res 15:133–137CrossRefGoogle Scholar
  30. Mohan BS, Hosetti BB (1999) Aquatic plants for toxicity assessment. Environ Res 81:259–274CrossRefPubMedGoogle Scholar
  31. Newmaster SJ, Bell FW (2002) The effects of silvicultural disturbances on cryptogam diversity in the boreal-mixedwood forest. Can J For Res 32:38–51CrossRefGoogle Scholar
  32. OECD (2006), Test No. 208: Terrestrial Plant Test: Seedling Emergence and Seedling Growth Test, OECD Publishing, ParisGoogle Scholar
  33. OECD (2006), Test No. 227: Terrestrial Plant Test: Vegetative Vigour Test, OECD Publishing, ParisGoogle Scholar
  34. OECD (2007) Section 2 – effects on biotic systems (draft). In: Guidelines for the testing of chemicals. Organisation for Economic Co-Operation and Development, ParisGoogle Scholar
  35. Quintanilla LG, Escudero A (2006) Spore fitness components do not differ between diploid and allotetraploid species of Dryopteris (Dryopteridaceae). Ann Bot 98:609–618CrossRefPubMedPubMedCentralGoogle Scholar
  36. Rodriguez-Gil JL, Catala M, Alonso SG, Maroto RR, Valcarcel Y, Segura Y, Molina R, Melero JA, Martinez F (2010) Heterogeneous photo-Fenton treatment for the reduction of pharmaceutical contamination in Madrid rivers and ecotoxicological evaluation by a miniaturized fern spores bioassay. Chemosphere 181:41–49Google Scholar
  37. Rodriguez-Gil JL, San Sebastián Sauto J, González-Alonso S, Sánchez Sánchez P, Valcarcel Y, Catalá M (2013) Development of cost-effective strategies for environmental monitoring of irrigated areas in Mediterranean regions: traditional and new approaches in a changing world. Agric Ecosyst Environ 181:41–49CrossRefGoogle Scholar
  38. Rowntree JK, Sheffield E (2005) The effects of Asulam spraying on non-target ferns. Canadian Journal of Botany, 83:1622–1629Google Scholar
  39. USEPA (1997) Terms of environment: glossary, abbreviations and acronyms (EPA publication no.175-B-97-001). U.S. Environmental Protection Agency, Washington DCGoogle Scholar
  40. USEPA (2002) Method 1003.0: Green Alga, Selenastrum capricornutum, growth test; chronic toxicityGoogle Scholar
  41. USEPA (2012) Series 850 – ecological effects test guidelines –terrestrial plants field study. U.S. Environmental Protection Agency, Office of Chemical Safety and Pollution Prevention (OCSPP), Washington DCGoogle Scholar
  42. Wang WC (1991) Literature-review on higher-plants for toxicity testing. Water Air Soil Poll 59:381–400CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Helena García-Cortés
    • 1
  • Myriam Catalá
    • 2
  • José Luis Rodríguez-Gil
    • 3
  1. 1.National Center for Environmental Health, National Health Institute Carlos IIIMadridSpain
  2. 2.Department of Biology and Geology, Physics and Inorganic ChemistryESCET, Rey Juan Carlos UniversityMadridSpain
  3. 3.Department of BiologyUniversity of OttawaOttawaCanada

Personalised recommendations