An in vitro submitochondrial bioassay for predicting acute toxicity in fish

A new, rapid, and reliable bioassay for monitoring the acute toxicity of aquatic samples has been developed. The method is based on metabolic changes in preparations made from isolated animal mitochondria which affect theratio of reduced to oxidized nicotinamide adenine dinucleotide (NADH/NAD) as measured in a spectrophotometer. Stable submitochondrial electron-transfer particles (SMP), made by sonic disruption of the cristae membranes of bovine heart mitochondria, afford the most practical biological test material because these vesicles can be stored indefinitely prior to use as frozen or freeze-dried preparations with full retention of biological activity. Under appropriate conditions, SMP can be programed to reduce exogenous NAD + by coupled reverse electron transfer (RET) or to oxidize NADH by the conventional electron transfer pathway (ETR). Exposure to toxicants inhibits one or both redox activities by an amount proportional to the toxicant concentration. Hence, bioassay procedures can be based on RET and ETR tests which are often complementary. Reliable data can be obtained in 2 to 18 min without interference caused by "hard water" conditions. The toxicities of 64 industrial chemicals, inorganics, and pesticides to SMP correlate closely with the results from tests with the fathead minnow (r 2 = 83%). The correlation was substantially poorer between fish toxicity and available data for the same toxicants from another short-term bioassay utilizing the Microtox procedure. In the new bioassay, the biological materials of the SMP test are handled like a chemical reagent. The test requires only small sample volumes, has good reproducibility, and promises to be relatively inexpensive, rapid, and simple. Bioassay variants and potential enhancements of the SMP test system are discussed.