Standardizing the Methodology of Sperm Cell Test with Paracentrotus Lividus
The sperm cell toxicity test with Paracentrotus lividus is widely used in Italy to assess the quality of complex aqueous matrices, but at present there is a shortage of standardized methodologies for the bioassay performance. In this work several critical points were considered, in order to verify the reliability of this bioassay and to improve its standardization.
In particular, we have studied the differences in EC50 values at different sperm: egg ratios and with different quantities of gametes; the influence of sperm and eggs on fertilization rate and the influence of different recipients materials (plastic or glass). At the same time, it was evaluated if the obtained EC50 values were in accordance with those reported in literature.
Experiments performed yielded EC50 values for copper ranging from 16.54 μ g/l to 69.59 μ g/l, with a total mean value ± SD of 39.79 μ g/l ± 11.17. These data fit with those found in literature for other echinoid species. According to the same authors, poor fertilization was probably due to the effects on sperm and not on eggs, and moreover the linear regression analysis performed on each experiment suggested that the best amount of eggs to use in the test was 1000.
Results from the comparison between the performance of the test in plastic or glass tubes showed an higher success in fertilization rate using borosilicate glass tubes.
Keywordscopper gametes Paracentrotus lividus plastic or glass tubes sea urchins sperm cell test sperm: egg ratio standardizing
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- ASTM: 1995, ‘Standard guide for conducting static acute toxicity tests with echinoid embryos’, E 1563–95, American Society for Testing and Materials, Philadelphia, pp. 1029–1046.Google Scholar
- ASTM: 2004, ‘Standard guide for conducting static acute toxicity tests with echinoid embryos’, E 1563–98, American Society for Testing and Materials, Philadelphia.Google Scholar
- Bay, S., Burgess, R. and Nacci, D.: 1993, ‘Status and applications of Echinoid (Phylum Echinodermata) toxicity test methods’, in: W.G. Landis, J.S. Hughes and M.A. Lewis (eds), Environmental Toxicology and Risk Assessment, ASTM SRP 1179, Philadelphia, pp. 281–302.Google Scholar
- Chapman, G.A., Denton, D.L. and Lazorchak, J.M.: 1995, ‘Short-term methods for estimating the chronic toxicity of effluents and receiving water to West Coast marine and estuarine organisms’, U.S. EPA, 600/R-95/136, Cincinnati, Ohio.Google Scholar
- Chia, F.-S. and Bickell, L.R.: 1983, ‘Echinodermata’, in: K.G. Adiyodi and R.G Adiyodi (eds), Reproductive Biology of Invertebrates, Volume II: Spermatogenesis and Sperm Function, John Wiley and Sons, New York, pp. 545–620.Google Scholar
- Decreto legislativo nr 152: 1999, ‘Disposizione sulla tutela delle acque dell'inquinamento e recepimento della direttiva 91/271/CEE concernente il trattamento delle acque reflue e della direttiva 91/676/CEE relativa alla protezione delle acque dell'inquinamento provocato dai nitrati provenienti da fonti agricole’, Gazzetta Ufficiale, 29 Maggio 1999, pp. 1–125.Google Scholar
- Dinnel, P.A.: 1991, ‘Proposed standard EXXX guide for conducting static acute toxicity tests with echinoid embryos’, in: Annual book of ASTM standards, Draft 1, pp. 1–45.Google Scholar
- Dinnel, P.A., Stober, Q.J., Crumley, S.C. and Nakatani, R.E.: 1982, ‘Development of a sperm cell toxicity test for marine waters’, in: J.G. Pearson, R.B. Foster and W.E. Bishop (eds.), Aquatic toxicology and hazard assessment, Fifth Conference, ASTM STP 766, Philadelphia, pp. 82–98.Google Scholar
- Environment Canada: 1991, ‘Fertilization assay with echinoids (sea urchin and sand dollars)’, Environmental Protection Series,EPS/1/RM, Ottawa, Canada.Google Scholar
- Environment Canada: 1992, ‘Biological test method: fertilization assay using Echinoids (sea urchins and sand dollars)’, Environmental Protection Series, EPS 1/RM/27, Ottawa, Canada.Google Scholar
- Finney, D.J.: 1971, Probit Analysis, Cambridge University Press, London, UK.Google Scholar
- Giambartolomei, F.M.: 1990, ‘Nuove acquisizioni sul test di fertilitá dello sperma di riccio di mare Paracentrotus lividus’, Thesis, University of Padova, Italy.Google Scholar
- Giuliani, S., Ennas, C., Pellegrini, D. and Kozinkova, L.: 2002, ‘The bioassay with sea urchin Paracentrotus lividus: different end-points to be applied to different environmental studies’, Fres. Environ. Bull. 11(10), 806–809.Google Scholar
- Giuliani, S., Lera, S., Kozinkova, L., De Ranieri, S. and Pellegrini, D.: 2004, ‘Sensibilitá del saggio di fecondazione con gameti di Paracentrotus lividus(L.) al variare di alcune condizioni sperimentali’, Biol. Mar. Medit.11(2), 330–333.Google Scholar
- Klemm, D.J., Morrison, G., Nurberg-King, T., Peltier, W. and Heber, M.: 1994, 'Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine and estuarine organisms’, U.S. EPA, 600-4-91-003, Cincinnati, Ohio.Google Scholar
- Kobayashi, N.: 1984, ‘Marine ecotoxicological testing with echinoderms’, in: G. Persoone, E. Jaspers and C. Claus (eds.), Ecotoxicological Testing for the Marine Environment, State Univ. Ghent and Inst. Mar. Scient. Res., Bredene, Belgium, pp. 341–405.Google Scholar
- Kozinkova, L., Ennas, C., Giuliani, S. and Pellegrini, D.: 2003, ‘Valutazione della qualitá dei gameti di Paracentrotus lividus (Lamarck) utilizzando Cu(NO_3)_2.3H_2O come tossico di riferimento su animali stabulati e non stabulati’, Biol. Mar. Medit. 10(2): 1101–1104.Google Scholar
- Lera, S. and Pellegrini, D.: 2004, ‘Evaluation of the fertilization capability of Paracentrotus lividussea urchin storaged gametes by the exposure to different aqueous matrices’, Environ. Monit. Assess.in press.Google Scholar
- Levitan, D.R., Sewell, M.A. and Chia, F.-S.: 1991, ‘Kinetics of fertilization in the sea urchin Strongylocentrotus franciscanus: interaction of gamete dilution, age, and contact time’, Biol. Bull.181, 371–378.Google Scholar
- Lillie, F.R.: 1915, ‘Studies of fertilization. VII. Analysis of variations in the fertilization power of sperm suspensions of Arbacia’, Biol. Bull. 28, 229–251.Google Scholar
- Morrison, G., Torello, E., Comoleo, R., Walsh, R., Kuhn, A., Burgess, R., Tagliabue, M. and Greene, W.: 1989, ‘Intralaboratory precision of saltwater short-term chronic toxicity tests’, J. Water Poll. Control Fed. 61, 1707–1710.Google Scholar
- Nacci, D., Jackim, E. and Walsh, R.: 1986, ‘Comparative evaluation of three rapid marine toxicity tests: sea urchin early embryo growth test, sea urchin sperm cell toxicity test and Microtox’, Environ. Toxicol. Chem. 5, 521–525.Google Scholar
- Nacci, D., Comoleo, P., Petrocelli, E. and Jackim, E.: 1991, ‘Earl-standard operating procedure conducting the sea urchin Arbacia punctulatafertilization test’, U.S. EPA, Environmental Research Laboratory, Narragansett, RI, pp. 125–131.Google Scholar
- Pennington, J.T.: 1985, ‘The ecology of fertilization of echinoid eggs: the consequences of sperm dilution, adylt aggregation, and synchronous spawning’, Biol. Bull. 169, 417–430.Google Scholar
- Tyler, A.: 1949, ‘A simple non-injurious method for inducing repeated spawning of sea urchins and sand dollars’, Collecting Net 19, 19–20.Google Scholar