Chronic effect and no effect concentrations (28 day) and acute toxicity (48 hr, LC50 and EC50) values were determined for Daphnia magna with some chlorinated benzenes, chlorinated ethanes, and tetrachloroethylene. Acute and chronic toxicity generally increased with the degree of chlorine substitution with these chemicals. The 48 hr LC50 values for hexachloroethane, pentachloroethane, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, 1,2,4-trichlorobenzene, 1,3-dichlorobenzene, and tetrachloroethylene were 2.9, 7.3, 62, 190, 270, 2.1, 7.4, and 18 mg/L, respectively. The lowest observable effect concentrations (LOEC) based on either reproductive impairment or growth for 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, 1,2-dichloroethane, 1,2,4trichlorobenzene, 1,3-dichlorobenzene, and tetrachloroethylene were 14, 26, 20, 0.69, 1.5, and 1.I mg/L, respectively. Length of animals at the end of chronic exposure was at least as sensitive a measure of toxic effect as reproduction with every chemical tested except 1,2-dichloroethane. The acute-chronic ratio ranged from 3 to 16 with these chemicals. The U.S. Environmental Protection Agency (EPA) has listed 129 toxic substances (priority pollutants) for immediate hazard assessment . These compounds were selected on the basis of known human or animal toxic and carcinogenic effects as well as on known or suspected presence in the environment. As part of this hazard assessment, toxicity tests are required with several groups of aquatic organisms. The present study was designed to examine the effects of acute and chronic exposure to chlorinated hydrocarbons, all of which are widely used industrial chemicals, on the cladoceran Daphnia magna. Of concern were two classes of chemicals, the monocyclic aromatics and the halogenated aliphatics. The monocyclic aromatics (1,3-dichlorobenzene and 1,2,4-trichlorobenzene) were selected because of immediate toxicity, persistence, and ability to bioaccumulate to some degree (Chapman et al. 1982). The halogenated aliphatics (1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane, pentachlorethane, hexachloroethane, and tetrachloroethylene) were also selected because of direct toxicity and also because they have been detected in aquatic organisms (U.S. EPA 1980b,c). In addition, the chlorinated ethanes form azeotropes in water, a property which may influence their persistence (Kirk and Othmer, 1963). Very little toxicity data are available for these compounds with Daphnia magna. Acute toxicities based on nominal concentrations for all 129 priority pollutants were determined for Daphnia magna by LeBlanc (1980). Otherwise, of the compounds tested in this study, only 1,1,2-trichloroethane has been tested for chronic toxicity (Adema 1978). Materials and Methods t Present address: Environmental Research Laboratory-Duluth, U.S. Environmental Protection Agency, 6201 Congdon Blvd., Duluth, MN 55804 Biological Methods Adult daphnids (Daphnia magna) were obtained from laboratory stock reared at the U.S. Environmental Protection Agency, Du680 J . E . Richter et al. luth, MN. Culturing and testing were done with Lake Superior water which was passed through a 5 micron fiber filter, heated to 20~ and aerated with filtered air. Culturing and testing systems were maintained in an enclosed constant temperature water bath (20 _+ 1~ A combination of Gro-Lux and Duro-Test (Optima FS) fluorescent bulbs provided 344 lumens at the air water interface and were on a 16L:8D photoperiod coupled with a 15 min transition period between light and dark phases. Brood cultures of 25 animals in 1-L beakers were maintained by renewing food (30 mg/L dry wt.), a slurry of trout chow and yeast, and water three times each week. For acute and chronic testing, first instar daphnids less than 24 hr old were collected from brood animals approximately three weeks old. Acute Bioassays: Acute bioassays were conducted according to ASTM (1980). To determine if feeding during these tests would influence toxicity, simultaneous experiments were conducted on fed and unfed animals. Data presented by Adema (1978) for four organic compounds revealed no clear-cut effects of the presence or absence of food on the derived LC50 values. Test containers were 200 ml Erlenmeyer flasks filled to 200 or 160 ml for unfed and fed tests, respectively. The food concentration was 20 rag/ L dry wt. (trout chow and yeast). The flasks were stoppered with foil wrapped, neoprene stoppers. Four replicates with five animals each were used for the control and six toxicant levels. Each toxicant concentration is ~60% of the next higher one. Acute toxicity values calculated were the 48 hr median effective concentration (EC50) based on complete immobilization, and the 48 hr median lethal concentration (LC50) based on death as defined by cessation of heart beat and gut movement. Because only careful microscopic examination can determine live from dead animals in a comatose state (Crosby et al. 1966), immobilization and death were determined with a 30 • dissection scope at the end of the exposure. EC50 and LC50 values were derived by the measured mean toxicant concentrations (average of initial and final test solution concentrations) and were calculated by probit, moving average, or binomial formulas depending on the characteristics of the data (Stephan 1977). In those cases where the data could be analyzed by more than one of these formulas, the results were never significantly different. Chronic Bioassays: Chronic 28 day bioassays were conducted according to ASTM (Proposed standard practice for conducting renewal life cycle toxicity tests with the daphnid Daphnia magna) Draft No. 4 (Comotto 1978), with minor modifications to control volatile chemical losses. Test containers were 200 ml Erlenmeyer flasks filled to 160 ml, with the exception of the test conducted with tetrachloroethylene in which flasks were filled to 175 ml of solution to reduce volatilization. Each of 7-10 replicate flasks at six test concentrations (geometric series with a 0.5 dilution factor) Table 3, contained one daphnid. The flasks were stoppered with foil wrapped, neoprene stoppers. Toxicant and food solutions (20 mg/L trout chow and yeast), were renewed three times each week. Young daphnids were filtered from each flask after transfer of the adults, washed onto a watch glass and counted alive with an Artek Counter ~ . If less than 20 animals were present, they were counted visually. An analysis of counting error was made for the Artek Counter against actual hand counts during several chronic tests. Standard deviations from actual counts were ~ l. 1 animals for any size sample counted. Live counts eliminated the additional steps of poisoning and stirring to redisperse them as described by LeBlanc (1979) and allowed the determination of live from dead animals. Chronic toxicity was determined by reproductive success and length of animals surviving the 28 days test. These data were Table 1. Chemical characterist ics (~g/L) of Lake Superior water a No. Chemical Mean Range Samples Chloride 1 , 2 1 7 1,170-1,340 18 Sodium 1,130 1,090-1,190 23 Calcium 13 ,695 13,000-14,700 23 Magnesium 3,123 2,940-3,590 23 Potassium 534 480-590 23 Strontium 16 12-27 10