Introduction

Since the aquatic environments such as coastal regions of marine area, lakes, rivers, etc., are burdened with thousands of pollutants, it is important to pay attention to their toxicities against the organisms in those ecosystems. Among these pollutants, we cannot turn a blind eye to the role of antifouling chemicals, which have been developed and used widely in the world. Fundamentally, most of them are highly toxic to organisms from both chronic and acute aspects, and thereby they function to control the adhesion and growth of various types of organisms on submerged structures of ships' hulls. Through human activities such as agriculture, aquaculture, or sailing of vessels around the world, these chemicals are discharged into the natural environment.

Under the direction of International Maritime Organization since 2003, organotin-based antifoulants, which have been most widely used mainly as antifoul-ing paints for many years, have been strictly regulated and their use was prohibited because of their severe negative effects on organisms. The ecotoxicological behaviors of new antifoulants in place of organotin compounds, however, have been insignificantly understood. Until recently, the reports on the toxicity of chemicals have been limited to those of each single chemical, but actually we frequently encounter the mixed state of chemicals rather than the single form -for instance, paint products are manufactured by mixing different kinds of antifoulants. Thus, it seems to be indispensable to estimate and evaluate the toxicities in the mixed state of these chemicals as fast and as quantitatively as possible.

Numerous types of organisms have been used in the different bioassay systems for the detection of toxicities. Among these assays, the usage of bioluminescent bacterium was proposed as a relatively simple method based on the reduction of bioluminescence intensity (BLI) by toxic compounds. Symbiotic bacterium Vibrio fischeri, which lives in the light organs of fish of the family Monocentridae as well as in the cephalopods Sepiola and Euprymna, is highly sensitive to environmental changes. In concrete terms, BLI changes of cells are strictly related to the cellular activities which ensure to reflect the certain level of contamination of the surrounding environment. An assay system using the fresh cells of V. fischeri was proposed in the previous studies of the authors, which showed fairly good response to the toxicity level of the samples tested.

The difference of toxicities between single chemicals and their mixture has been attributed to the interaction effects, which leads to the classification into five typical patterns as follows: antagonism, no addition, partial addition, concentration addition, and supra-addition. In this regard, the highest toxicity and sum of all toxicities (ST) are the important critical indices to classify the interaction effects. In general, toxicity of each chemical has been expressed in terms of toxicity unit (TU), which was defined as the ratio of the concentration of each chemical to the respective EC50 value. Actual toxicity unit (ATU) for a mixture was the percentage after division by 50% for the data such as cell viability or growth inhibition. To clarify the interaction effects, total theoretical toxicity unit (TTTU) corresponding to ST was calculated from each chemical in the mixture and compared with ATU. If ATU is less than TTTU, the interaction effect was defined as antagonism. Using the same way for analyses, each combination is reasonably categorized as follows: if ATU is equal to or higher than TTTU, they are designated to the concentration addition or synergism, respectively. Based on the methodology mentioned above, Konemann proposed mixture toxicity indices (MTIs) as a quantitative indicator for the toxicity in mixed chemicals. Calculations of MTI values enable us to estimate the extent of toxicity enhancement or reduction by mixing the single chemicals together. In the case of samples showing the synergistic effect (MTI > 1), the relatively higher the values of MTI are, the more serious is the enhancement of toxicity. In the antagonistic effect, on the contrary, the relatively lower the values of MTI are, the more serious is the reduction of toxicity.

In this section, the authors demonstrate the evaluation data obtained by their assay system for 11 different kinds of single antifouling chemicals as well as 45 combinations composed of two of them. In addition, the interactions of these chemicals were examined based on the EC50 values as well as the percentage of inhibition efficiency (INH, %) for single and mixed chemicals, the latter of which were also analyzed through MTI calculations to classify the effects by mixing the chemicals.

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