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API DR79 1984 Edition, January 1, 1984
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Development of a Diluting Concentration method for Testing the Acute Toxicity of Chemically Dispersed Oil, and the Results of Comparative studies
Additional Comments: W/D NO S/S * REPRINTED IN 1993
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Introduction

For several years the accepted method of testing the comparative toxicity of pollutants for a range of freshwater and marine species has been the 96-hr LC50 method (APHA, 1975). These tests were usually conducted in closed ("static") systems without feeding or replenishment of toxicants. The 4-day period was such that feeding was not needed (in most cases) and many of the toxicants did not evaporate substantially. As more chemical analyses were conducted, investigators began to recognize that significant amounts of many toxicants sorbed to the walls of vessels, evaporated, and were taken up by the organisms. In tests with petroleum, there were also problems with droplets moving to the surface (forming slicks) and numerous alterations related to different specific components.

The inconsistencies were such that only water-soluble (or accommodated) fractions of oil could be linearly diluted. Decreases in component concentrations in as little as 24 hr could be as high as 90%. It was clearly necessary to utilize flowing exposures in tests with oils, where the dominant hydrocarbons were still those that dissolved in seawater. Tests conducted with flowing exposures of soluble hydrocarbons have, not suprisingly, shown that organisms are affected at lower concentrations than those produced in static tests (Vanderhorst et al., 1976). Some of the flowing exposures have been conducted for several days or even weeks (Anderson et al., 1980). Many sublethal studies were also conducted for weeks or months and indicated that quite low concentrations affected the organisms (Caldwell et al., 1977). It became apparent that there was a need to develop a system for comparing the long-term tests with short-term (96-hr) bioassays. The most logical means of making such comparisons would be to consider time of exposure as a variable 2 equal in importance to concentration of exposure. If this hypothesis were valid, then a 2-day exposure to 10 parts per million (ppm) of a water-soluble extract should produce the same effect as a 5-day exposure to 4 ppm, or a 10-day exposure at 2 ppm, etc. The relationship has been found to be consistent for exposure periods between about 8 hours and 8 days. Anderson et al. (1980) have shown that three species of crustaceans (2 shrimp and a mysid) exhibited mortality in a flowing exposure to an extract of Prudhoe Bay crude oil which was a function of the product of concentration and time. The product was referred to as the "toxicity index" (in ppm-days). When 50% mortality occurred in a tank, the time in days was merely multiplied by the mean exposure concentration, which was measured by infrared. Spectrophotometry (IR) at 1 or 2 day intervals. All 50% mortality data could be plotted on log-log paper with time versus concentration, and a curve of -1.2 slope was produced. The slopes did not vary with the species, but the most sensitive species (a mysid) produced a curve which indicated greater sensitivity than the two shrimp species. Since the toxicity index has proven to be a valid approach for estimating the toxicity of soluble hydrocarbons, we proposed to apply the same methodology to studies on chemically dispersed oil. In addition to toxicity measurements for oil dispersions, it was necessary to consider thephysical form of the exposure materials.