Microtox test

Kamlet, M. J., Doherty, R. M., Veith, G. D., Taft, R. W., Abraham, M. H. (1986) Solubility properties in polymers and biological media. 7. An analysis toxicant properties that influence inhibition of bioluminescence in Photobacterium phosphoreum (the Microtox test). Environ. Sci. Technol. 20, 690-695. 

The first steps in bypassing of the biological, technological, and financial burden of live stock culturing or maintenance were made more than 20 years ago through the development of a bacterial luminescence inhibition test [34,35] this bioassay is presently known and used worldwide as the Microtox test. The revolutionary principle of this test is that it uses a lyophihzed strain of a (marine) bacterium Photobacterium phosphoreum). This makes the bioassay apphcable anytime, anywhere, without the need for continuous culturing of the test species. 

The Microtox test has been used for determination of toxicity of wastewater effluents, complex industrial wastes (oil refineries, pulp and paper), fossil fuel process water, sediments extracts, sanitary landfill, and hazard waste leachates [19]. 

In France, industrial effluents are regularly monitored for toxicity with daphnids. The toxicity data are used as a base for discharge taxation [193]. The Microtox test, chronic toxicity test, and a test on mutagenicity to the set of required bio-criteria are also used for wastewater monitoring [12,194]. 

Bogaerts, P. Bohatier, J. Bonnemoy, E. Use of the ciliated protozoan Tetrahymena pyriformis for the assessment of the toxicity and quantitative structure-activity relationships of xenobiotics comparison with the microtox test. Ecotox. Environ. Safe. 2001, 49, 293-301. 

The bacterial Microtox tests and the other in vitro bioassays clearly indicated differences in sediment toxicity between locations (Table 7). In the Microtox SP assay inhibitory effects were found in sediment extracts from the Port of Amsterdam transect (i.e. TU values greater than 20 at sites 7 and 11). The highest response in the Mutatox assay was found at Oranjesluis (site 11) in the Port of Amsterdam. The reference values from the Mutatox assay at this site were below... 

Table 7 Results of Microtox tests and in vitro bioassays. ...

Bennett, J. and Cubbage, J. (1992b) Review and evaluation of Microtox test for freshwater sediments, Washington State Department of Ecology, Washington, 28 pp. 

Boluda, R., Quintanilla, J.F., Bonilla, J.A., Saez, E. and Gamon, M. (2002) Application of the Microtox test and pollution indices to the study of water toxicity in the Albufera Natural Park (Valencia, Spain),... 

Bonnemoy, F., Lavedrine, B. and Boulkamh, A. (2004) Influence of UV irradiation on the toxicity of phenylurea herbicides using Microtox test, Chemosphere 54 (8), 1183-1187. 

Dieter, C.D., Hamilton, S.J., Duffy, W.G. and Flake, L.D. (1994) Evaluation of the Microtox test to detect phorate contamination in wetlands, Journal of Freshwater Ecology 9 (4), 271-280. 

Pardos, M., Benninghoff, C., Gueguen, C., Thomas, R., Dobrowolski, J. and Dominik, J. (1999a) Acute toxicity assessment of Polish (waste)water with a microplate-based Hydra attenuata assay a comparison with the Microtox test, The Science of The Total Environment 243-244, 141-148. 

Redondo, M.J., Lopez-Jaramillo, L., Ruiz, M.J. and Font, G. (1996) Toxicity assessment using the microtox test and determination of pesticides in soil and water samples by chromatographic techniques, Toxicology Letters 88 (Supplement 1), 30-30. 

Tarkpea, M. and Hansson, M. (1989) Comparison between two Microtox test procedures, Ecotoxicology and Environmental Safety 18 (2), 204-210. 

Among the two genotoxicity tests, the Mutatox is a commercial test using a bacterial strain and is no more difficult to perform than the well known Microtox test. The test on induction of micronuclei in erythrocytes of Xenopus laevis larvae developed by van Hummelen et al. (1989) requires specialized skills and training and is routinely performed only in a few laboratories. However, this test is currently being standardized by ISO (International Standardization Organisation). 

Microbiological tests such as the Microtox test report a non-quantal endpoint, which is an IC50. ECs are for quantal data (e.g., of immobilised daphnids). 

With respect to leachate pre-treatment, different methods have been reported in the literature involving steps such as decantation, centrifugation, filtration and pH adjustment. Specific pre-treatment may well have some influence on chemical or ecotoxicological results (Isidori et ah, 2003). Moreover, some bioassays can only be run on 0.45 pm filtered leachates (e.g. Ames test) or those having undergone pH adjustment (e.g., Microtox test). 

Microtox test) Vibrio fisheri ISO 11348-3 (1999) Inhibition of bioluminescence Acute... 

Table 5 summarizes pH, conductivity and ecotoxicological results obtained for each waste and each L/S ratio. For the Microtox test, a preliminary study (results not shown) had demonstrated that a 30-min exposure time was sufficient for testing 2SL leachates while 60 min was a more optimal time for testing the BA leachates. pH and conductivity values, obtained after water extraction (L/S of 2 and 10) of both wastes, were relatively high. Toxicity responses obtained from BA leachates varied from 4.2 TU (ceriodaphnid test, L/S 2) to 65.3 TU (algal test, L/S 2), whereas they... 

In contrast, the decreasing sensitivity sequence for the 2SL leachates was the following Microtox test > ceriodaphnid test > algal test daphnid test > Mutatox test. This indicates that the Microtox test should be the bioassay of choice to assess 2SL toxicity fluxes in the WASTOXHAS procedure. 

Results of the ecotoxic potential of leachate fluxes from BA waste are presented in Figure 7. Similarly results for the 2SL waste are presented in Figure 8. For each waste tested, bioassay responses showed adequate repeatability for the three replicate extraction columns (n = 3) employed. Indeed, averaged coefficients of variation determined with the Microtox test and the algal test were respectively 14.1% and 15.6% for columns filled with BA waste, and 19.4% and 27.3%, respectively, for columns filled with 2SL waste. 

Figure 7. Downward-flow column study - Toxicity responses of the Microtox test and the algal test on BA leachates in relation to the liquid-to-solid ratio (L/S).

As expected, based on the prerequisite study, algae were more sensitive to tested fractions of BA leachates than bacteria (Microtox test). In contrast, bacteria proved to be more sensitive to most of 2SL leachate fractions, although algae were more sensitive to the L/S 6 and L/S 8 fractions. 

Based on the different results obtained for the algal and Microtox tests (Figures 7 and 8), a waste PEEP index value was calculated for each waste and each L/S ratio assessed. Each waste index value was then plotted as a function of the corresponding L/S ratio (Fig. 9) and a simple non-linear regression fit (Power model, y = axb) was applied to predict the ecotoxicological hazard potential of leachate fluxes between L/S 4 and L/S 30 ratios. 

Table 9 gives an overview of results obtained from the column tests and the field tests for BA and 2SL wastes up to an L/S ratio of 2 and an L/S ratio of 8, respectively. This outlook allows a comparison of the two procedures based on the sensitivity responses of the algal and Microtox tests and on the waste PEEP index values for L/S ratios experimented in the field. For the purposes of this comparison, waste PEEP index values for the field percolates were recalculated using results of both tests presented in Table 8. 

In the Microtox test, it is a standard culture of freeze-dried (lyophilized) Vibrio fischeri, stored in small, sealed vials which each contain about 100 million cells. Volume 1(1,2). 

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