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Order of appearence	Full citation	SRCosmos Link
1	US EPA (1993) ‘Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms’, US Environmental Protection Agency EPA/600/4-90/027F, Fourth Edition.
2	Microbics Corporation (1992) ‘Microtox Manual’. AZUR Environmental, Carlsbad, CA.
3	Fulladosa E, Murat JC, Martinez M, Villaescusa I, (2005) ‘Patterns of metals and arsenic poisoning in Vibrio fischeri bacteria’ Chemosphere, (in press).
4	Kaiser KLE, Palabrica VS, (1991) ‘Photobacterium phosphoreum Toxicity Data Index’ Wat. Pollut. Res. J. Canada 26 (3), 361-431.
5	Davoren M, Fogarty AM, (2004) ‘A test battery for the ecotoxicological evaluation of the agri-chemical Environ’ Ecotox. Environ. Safe., 59, 116–122.
6	Gutierrez M, Etxebarria J, De-Las-fuentes L, (2002) ‘Evaluation of wastewater toxicity: comparative study between Microtox and activated sludge oxygen uptake inhibition’ Water Res., 36, 919–924.
7	Fulladosa E, Murat JC, Villaescusa I, (2005) ‘Study on the toxicity of binary equitoxic mixtures of metals using the luminescent bacteria Vibrio fischeri as a biological target’ Chemosphere, 58, 551–557.
8	Mowat FS, Bundy KJ, (2002). ‘Experimental and mathematical/computational assessment of the acute toxicity of chemical mixtures from the Microtox assay’ Adv. Environ. Res., 6, 547–558.
9	Villaescusa I, Matas C, Hosta C, Martinez M, Murat JC, (1998) ‘Evaluation of lead(II) and nickel(II) toxicity in NaCl and NaClO4 solutions by using Microtox bioassay’ Fresenius J. Anal. Chem., 361, 355–358.
10	Utgikar VP, Chaudhary N, Koeniger A, Tabak HH, Haines JR, Govind R, (2004) ‘Toxicity of metals and metal mixtures: analysis of concentration and time dependence for zinc and copper’ Water Res., 38, 3651– 3658.
11	Deheyn DD, Bencheikh-Latmani R, Latz MI, (2004) ‘Chemical Speciation and Toxicity of Metals Assessed by Three Bioluminescence-Based Assays Using Marine Organisms’ Environ. Toxicol., 19, 161–178.
12	Ruiz MJ, Lopez-Jaramillo L, Redondo MJ, Font G, (1997) ‘Toxicity Assessment of Pesticides Using the Microtox Test: Application to Environmental Samples’ Bull. Environ. Contam. Toxicol., 59, 619-625.
13	Choi K, Meier PG, (2001) ‘Toxicity Evaluation of Metal Plating Wastewater Employing the Microtox Assay: A Comparison with Cladocerans and Fish’ Environ. Toxicol., 16, 136-141, 2001.
14	Choi K, Sweet LI, Meier PG, Kim PG, (2004). ‘Aquatic Toxicity of Four Alkylphenols (3-tert-Butylphenol, 2-Isopropylphenol, 3-Isopropylphenol, and 4-Isopropylphenol) and Their Binary Mixtures to Microbes, Invertebrates, and Fish’ Environ. Toxicol. 19, 45–50.

Keywords	Heavy metals, organic pollutants, toxicity, Microtox, Vibrio fischeri
Abstract	Microtox acute toxicity test is widely used for the toxicity assessment of various environmental samples. This test is based on the measurement of bioluminescence inhibition of the bacteria Vibrio fischeri after sample exposure at various contact times. However, the response of test species is greatly affected by the type of the toxic compounds, resulting in variations in the appropriate exposure time. The aim of this study was the investigation of toxic effect of heavy metals and various organic compounds on V. fischeri, as a function of exposure time. The examined heavy metals were Cd, Cr, Cu, Hg and Ni, while the organic compounds included phenol, pentachlorophenol, benzene, orange II and lindane. The toxicity of each compound under various concentrations was examined at different exposure times and the corresponding response was measured. Toxicity increased significantly by exposure time for the heavy metals; the highest toxicity (the lowest EC50 value) was observed after 30 min exposure time. Furthermore Cd and Ni presented the highest increase of toxic effect on the test species by exposure time. The EC50 of Cd and Ni decreased from 50.1 and 74.5 mg/L after 5 min exposure time to 5.3 and 7.6 mg/L after 30 min exposure time, respectively. Hg indicated the lowest toxicity increase, with EC50 values ranking from 0.12 to 0.07 mg/L at exposure times from 5 to 30 min, respectively. However, the toxicity of organics compounds was only slightly affected by exposure time; for pentachlorophenol exposure time at 5 and 30 min the EC50 ranged from 1.81 to 1.20 mg/L, while for the other organic compounds the exposure time did not significantly influence the EC50 values. Although there is not a clear interpretation of the type of the chemicals that might affect the time-effect dependencies for Microtox test, it has been demonstrated in this work that the toxic effect of heavy metals on V. fischeri presented slow reaction rate with exposure time, while organic substances presented a fast toxic action, taking place in the first minutes of exposure. According to the current results, an extension of exposure time up to 30 min may be required depending upon the presence of specific compounds. The time effect dependency of Microtox test to the contaminant type could be a useful tool for the interpretation of toxicity results of an environmental sample regarding to the type of contaminants that may be present in the sample.
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Name	Affiliation	Home page	e-mail	Total pubs
Kungolos A	Department of Planning & Regional Development, University of Thessaly		kungolos@uth.gr	22
Kyriazis S				1
Petala M	Chemical Process Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki			7
Sakellaropoulos GP	Chemical Process Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki			18
Samaras P	Department of Food Technology, Alexander Technological Educational Institute of Thessaloniki, GR-57400 Thessaloniki, Greece		samaras@food.teithe.gr	35
Tsiridis V	Department of Planning and Regional Development, University of Thessaly, 38334, Volos, Greece			8