Development of a microalgal PAM test method for Cu(II) in waters: comparison of using spectrofluorometry


Test methods are needed to monitor Cu concentrations in reservoirs and water supplies. Dictyosphaerium chlorelloides (Chlorophyta) cells were immobilized in a silicate sol–gel and the toxic effects of Cu(II) were examined using different techniques: fluorescence measurements (using a spectrofluorometer with an optic fiber coupled to a flow cell or a 96-well-plate reader) or by Pulse Amplitude Modulation (PAM) parameters using a portable instrument and the pulse saturation method. Fm′ and qN were the most sensitive indicator parameters when performing Cu analysis in water. D. chlorelloides PAM biosensor presented a detection limit of 0.6 mg l−1 for Cu(II), within the limits to establish if Cu concentrations exceeded regulatory levels. Moreover, a 1.9 mg Cu l−1 (30 μM) resistant strain of the D. chlorelloides microalgae was produced in order to obtain more selectivity on the metal determination.

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Fig. 1
Fig. 2



Dictyosphaerium chlorelloides

EC50 :

Effective concentration causing a 50% of variation in the signal (mg l−1)


Fluorescence yield measured briefly before application of the last saturation pulse; normally measured in the presence of actinic light


Maximal fluorescence yield of a dark-adapted sample, with all PSII reaction centers fully closed


Maximal fluorescence yield reached during last saturation pulse with an illuminated sample


Minimal fluorescence yield of a dark-adapted sample, with all PSII reaction centers fully open


Minimal fluorescence yield of a preilluminated membrane containing the algae; determined briefly after turning off the actinic light and after several seconds of far-red illumination to assure full oxidation of PSII acceptors


Fv = Fm − Fo


Limit of detection (mg l−1)


Pulse Amplitude Modulation


Photosynthetically active radiation


Photosystem II


Coefficient of photochemical quenching defined by: qP = (Fm′ − F)/(Fm′ − Fo′). It may vary between 0 and 1


Coefficient of nonphotochemical quenching defined by: qN = (Fm − Fm′)/(Fm − Fo′). It may vary between 0 and 1


(=Φ′) Effective quantum yield of photochemical energy conversion at PSII reaction centers calculated according with: Y = (Fm′ − F)/Fm′


  1. Ait Ali N, Juneau P, Didur O, Perreault F, Popovic R (2008) Effect of dichromate on photosystem II activity in xanthophylls-deficient mutants of Chlamydomonas reinhardtii. Photosynth Res 95:45–53

    Article  CAS  Google Scholar 

  2. Altamirano M, García-Villada L, Agrelo M, Sánchez-Martín L, Martín-Otero L, Flores-Moya A, Rico M, López-Rodas V, Costas E (2004) A novel approach to improve specificity of algal biosensors using wild-type and resistant mutants: an application to detect TNT. Biosens Bioelectron 19:1319–1323

    Article  CAS  Google Scholar 

  3. Bengtson Nash SM, Quayle PA, Schreiber U, Muller JF (2005) The selection of a model microalgal species as biomaterial for a novel aquatic phytotoxicity assay. Aquat Toxicol 72:315–326

    Article  CAS  Google Scholar 

  4. Bhatia RP, Brinker CJ, Gupta AK, Singh AK (2000) Aqueous solgel process for protein encapsulation. Chem Mater 12:2434–2441

    Article  CAS  Google Scholar 

  5. Chaplen FWR, Vissvesvaran G, Henry EC, Jovanovic GN (2007) Improvement of bioactive compound classification through integration of orthogonal cell-based biosensing methods. Sensors 7:38–51

    Article  CAS  Google Scholar 

  6. Chen J, Xu Y, Xin J, Li S, Xia C, Cui J (2004) Efficient immobilization of whole cells of Methylomonas sp. strain GYJ3 by sol-gel entrapment. J Mol Catal B-Enzym 30:167–172

    Article  CAS  Google Scholar 

  7. Coiffier A, Coradin T, Roux C, Bouvet OMM, Livage J (2001) Sol-gel encapsulation of bacteria: a comparison between alkoxide and aqueous routes. J Mater Chem 11:2039–2044

    Article  CAS  Google Scholar 

  8. Costas E, López-Rodas V (2006) Copper sulphate and DCMU-herbicide treatments increase asymmetry between sister cells in the toxic cyanobacteria Microcystis aeruginosa: Implications for detecting environmental stress. Water Res 40:2447–2451

    Article  CAS  Google Scholar 

  9. Council Directive 98/83/EC of 3 November (1998) On the quality of water intended for human consumption. Official Journal of the European Communities L 330:32–54

    Google Scholar 

  10. García-Villada L, Rico M, Altamirano M, Sánchez-Martín L, López-Rodas V, Costas E (2004) Occurrence of copper resistant mutants in the toxic cyanobacteria Mycrocistis aeruginosa: characterisation and future implications in the use of copper sulphate as algaecide. Water Res 38:2207–2213

    Article  CAS  Google Scholar 

  11. Hameed MSA, Ebrahim OH (2007) Biotechnological potential uses of immobilized algae. J Agric Biol 9:183–192

    Google Scholar 

  12. Juneau P, Dewez D, Matsui S, Kim SG, Popovic R (2001) Evaluation of different algae species sensitivity to mercury and to metolachlor by PAM-fluorometry. Chemosphere 45:589–598

    Article  CAS  Google Scholar 

  13. Juneau P, El Berdey A, Popovic R (2002) PAM fluorometry in the determination of the sensitivity of Chlorella vulgaris, Selenastrum capricornutum, and Chlamydomonas reinhardtii to copper. Arch Environ Contamin Toxicol 42:155–164

    Article  CAS  Google Scholar 

  14. Juneau P, Green BR, Harrison PJ (2005) Simulation of Pulse-Amplitude-Modulated (PAM) fluorescence: limitations of some PAM-parameters in studying environmental stress effects. Photosynthetica 43:75–83

    Article  CAS  Google Scholar 

  15. Kukarskikh GP, Graevskaya EE, Krendeleva TE, Timofeev KN, Rubin AB (2003) Effect of methylmercury on the primary photosynthetic activity of green microalgae Chlamydomonas reinhardtii. Biofizika 48(5):853–859

    CAS  Google Scholar 

  16. Mallick N, Mohn FH (2003) Use of chlorophyll fluorescence in metal-stress research: a case study with the green microalga Scenedesmus. Ecotoxicol Environ Saf 55(1):64–69

    Article  CAS  Google Scholar 

  17. Miao AJ, Wang WX, Juneau P (2005) Comparison of Cd, Cu, and Zn toxic effects on four marine phytoplankton by pulse-amplitude-modulated fluorometry. Environ Toxicol Chem 24:2603–2611

    Article  CAS  Google Scholar 

  18. Nassif N, Roux C, Coradin T, Rager MN, Bouvet OMM, Livage J (2003) A sol-gel matrix to preserve the viability of encapsulated bacteria. J Mater Chem 13:203–208

    Article  CAS  Google Scholar 

  19. Nguyen-Ngoc H, Tran-Minh C (2007) Fluorescent biosensor using whole cells in an inorganic translucent matrix. Anal Chim Acta 583:161–165

    Article  CAS  Google Scholar 

  20. Peña-Vázquez E, Maneiro E, Pérez-Conde C, Moreno-Bondi MC, Costas E (2009) Microalgae fiber optic biosensors for herbicide monitoring using sol-gel technology. Biosens Bioelectron 24:3538–3543

    Article  CAS  Google Scholar 

  21. Podola B, Melkonian M (2005) Selective real-time herbicide monitoring by an array chip biosensor employing diverse microalgae. J Appl Phycol 17:261–271

    Article  CAS  Google Scholar 

  22. Ralph PJ, Smith RA, Macinnis-NG CMO, Seery CR (2007) Use of fluorescence-based ecotoxicological bioassays in monitoring toxicants and pollution in aquatic systems; review. Toxicol Environ Chem 89:589–607

    Google Scholar 

  23. REAL DECRETO 140/2003 de 7 de febrero (2003) Por el que se establecen los criterios sanitarios de la calidad del agua de consumo humano. BOE 45:7228–7245

    Google Scholar 

  24. REAL DECRETO 1744/2003 de 19 de diciembre (2003) por el que se modifica el Real Decreto 1074/2002, de 18 de octubre, por el que se regula el proceso de elaboración, circulación y comercio de aguas de bebida envasadas. BOE 312:46524–46529

    Google Scholar 

  25. Reboud X, Majerus N, Gasquez J, Powles S (2007) Chlamydomonas reinhardtii as a model system for pro-active herbicide resistance evolution research. Biol J Linn Soc 91:257–266

    Article  Google Scholar 

  26. Tibuzzi A, Rea G, Pezzotti G, Esposito D, Johanningmeier U (2007) A new miniaturized multiarray biosensor system for fluorescence detection. J Condens Matter 19:1–12

    Article  CAS  Google Scholar 

  27. Vinot I, Pihan JC (2005) Circulation of copper in the biotic compartments of a freshwater reservoir. Environ Pollut 133:169–182

    Article  CAS  Google Scholar 

  28. Wang WX, Dei RCH (2006) Metal stoichiometry in predicting Cd and Cu toxicity to a freshwater green alga Chlamydomonas reinhardtii. Environ Pollut 142:303–312

    Article  CAS  Google Scholar 

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This study received financial support from the Comunidad Autónoma de Madrid (Project S0505/AMB374), the ESF, the ERDF. Elena Peña Vázquez acknowledges the Isidro Parga Pondal Program (Xunta de Galicia) for her research grant in the Complutense University.

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Correspondence to E. Costas.

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Peña-Vázquez, E., Pérez-Conde, C., Costas, E. et al. Development of a microalgal PAM test method for Cu(II) in waters: comparison of using spectrofluorometry. Ecotoxicology 19, 1059–1065 (2010).

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  • Biosensor
  • Algae
  • Dictyosphaerium chlorelloides
  • PAM
  • Copper