Development of an invertebrate bioassay to screen petroleum refinery effluents discharged into freshwater

A simple method using unsophisticated equipment is suggested for on-site toxicity testing of refinery effluents. This method will provide an inexpensive means of identifying problem materials and establishing priorities for coping with these materials. An arbitrary reference mixture (ARM), containing six common constituents of refinery wastewaters, was used for static toxicity tests on 15 species of freshwater invertebrates and three species of fish. Daphnia pulex was selected for further testing because it was the most sensitive of the animals tested, relatively inexpensive and easy to maintain, and a potential fish-food organism. If the reference mixture were representative of a refinery effluent, the Daphnia bioassay would be sufficiently sensitive to give reliable results within 48 h, whereas a fish bioassay would show no toxicity even after 96 h.

To assess the suitability of the Daphnia bioassay, tests were conducted by personnel at six petroleum refineries. Duplicate tests were in agreement. Results of tests using actual refinery effluents ranged from no toxicity after 96 h to a mean lethal concentration of 1.2% effluent after 48 h. The data presented show that the method was reproducible and that refinery personnel were able to perform the bioassay. Potential problems and advantages of the method are discussed.

The test temperature is the most important environmental factor affecting Daphnia bioassay results. As temperature increased, so did toxicity. Light intensity and culture age or condition may affect bioassay results, but the reasons are unclear. The differences due to photoperiod are not significant as long as there is not continuous light or dark. Chemical features such as hardness and pH of the test affect bioassay results with Daphnia as they do for fish: as hardness increases, toxicity of the ARM decreases, and as pH increases, toxicity of ammonium chloride increases.

No. 2 fuel oil is the single most important toxic component of the ARM. Without the oil the ARM is 17 times less toxic to Daphnia. Emulsification of the ARM increases its toxicity. Because fuel oil is the dominant toxic component, it was not possible to accurately assess a change in toxicity when lead, zinc, or cyanide was added to the ARM.

Expressed as multiples of the ARM the 96-h LC50 values for fish were 1.7 for rainbow trout, 4.6 for brook stickleback, 14 for bluegill, 20 for goldfish and more than 32 for fathead minnow. The 24- and 48-h LC50 values for Daphnia pulex were 0.42 and 0.033, respectively.

Compared to fish, Daphnia gave a larger variation in response to ARM toxicity. The possibility that variation was due to changes in susceptibility during the molt cycle was tested. Animals of known molt-stage were exposed for two h to dilute potassium chromate (0.56 mg/1 Cr⁺⁶). Animals which molted during the exposure period showed a significantly higher mortality than those which did not molt. Individuals exposed continuously to dilute potassium chromate solutions (0.56 and 0.10 mg/1 Cr⁺⁶) exhibited a significantly lower mean survival time if molting occurred less than 4 h prior to the beginning of exposure. It is suggested that molting is a significant source of variability in Daphnia.