Effects of Diet on Seven-Day Ceriodaphnia dubia Toxicity Tests

The purpose of this study was to evaluate the effects of four diets on the results of seven-day Ceriodaphnia dubia toxicity tests. Survival and reproduction were used as indices to detect the sensitivity of this species to acute and chronic copper stress. All toxicity tests were conducted using the moderately hard reconstituted water recommended in 1989 by the U.S. Environmental Protection Agency. Diet differentially affected the acute and chronic toxicity of copper. Daphnids fed Selenastrum capricomutum (alga) showed the greatest sensitivity, followed by those fed the alga Chlamydomonas reinhardti, then by animals fed a Yeast-Cerophyll^-Trout Food (YCTF) mixture plus Selenastrum, and finally by animals fed YCTF alone. These differences may result from the poor nutritional adequacy of Selenastrum when fed alone, the different caloric contents of the diets, the increased toxicant uptake by the organisms through ingestion of copperladen algal cells, and/or copper ions sequestered by fats and insoluble substances in YCTF. We recognize that diet is an important variable in seven-day toxicity tests, and that the selection of a diet should not be based only on its effects on long-term culturing of C. dubia, but also on its possible effects on test results. OHIO J. SCI. 93 (3): 44-47,1993 INTRODUCTION Daphnids are frequently used as experimental organisms for evaluating the toxicity of complex effluents. Traditionally, bioassays using complete life cycles of Daphnia magna were widely used and, with few exceptions, are well standardized (APHA1989). However, because of their time-consuming (3-4 weeks) and costly nature, these tests are not practical for routine monitoring. To circumvent these problems the U.S. Environmental Protection Agency (U.S. EPA) introduced, in 1984, the seven-day static renewal survival and reproduction test using Ceriodaphnia dubia. The introduction of the seven-day toxicity test triggered an extensive use of C. dubia as an experimental animal (Mount and Norberg-King 1985, Mount et al. 1986, Knight and Waller 1987, Winner 1988). But unlike D. magna, C. dubia does not have a long history in routine testing. The first article on culture methods and description of the use of this species in toxicity testing was published in 1984 (Mount and Norberg 1984). As more researchers have adopted C. dubia as a test organism, periodic problems have been encountered in conducting valid toxicity tests, especially with regard to food type (DeGraeve and Cooney 1987). Consequently, test methods and approaches have been modified and investigators seem to show preferences for particular diets; therefore, many different diets have been proposed for C. dubia. The initial food described was a simple suspension of yeast (Mount and Norberg 1984). Several authors (Knight and Waller 1987, Cowgill et al. 1985a, Winner 1989) have suggested various species of algae for maintaining cultures of this cladoceran. A diet commonly used is a YeastCerophyllTM-Trout Food (YCTF) mixture which was suggested by investigators at the U.S. EPA in 1985 (Horning and Weber 1985). At present the U.S. EPA recommends a 'Manuscript received 22 June 1992 and in revised form 17 February 1993 (#92-18). feeding combination of YCTF and the alga Selenastrum capricomutum (U.S. EPA 1989). Although efforts have been made to evaluate the adequacy of several diets for maintaining C. dubia cultures (Cowgill et al. 1985a, Winner 1989, Norberg and Mount 1985), more work is needed to evaluate the effects that diet might have on the seven-day C. dubia toxicity test results. This article presents the evaluation of the effects of four diets on the sensitivity of C. dubia to copper in seven-day static renewal survival and reproduction toxicity tests. MATERIALS AND METHODS Seven-day toxicity tests were run for each of the diets evaluated in this study. All tests were initiated with young (<24 h old) C. dubia. Test organisms were maintained individually in 30 ml disposable clear plastic cups containing 15 ml of test solution. Trays holding the plastic cups were covered with clear plastic sheets to retard evaporation. Organisms were maintained in an environmental chamber at 23± l ° C o n a l 6 h light, 8 h dark photoperiod at a light intensity of approximately 70 ft-c. Animals were transferred to fresh test solution daily. Each daphnid was fed the appropriate diet and examined every day until it died (i.e., showed no movement) or until the test ended. Offspring were counted and discarded at renewal time. Records of survival, young per female, number of broods, and age at reproductive maturity were kept and used to estimate the endpoints for acute and chronic toxicity reported in this study. All tests were conducted using the moderately hard reconstituted water recommended by the U.S. EPA (1989) as the dilution water. Total hardness, alkalinity, and pH ranged from 80 to 90 mg CaCO/L, 55 to 70 mg CaCO/L, and 7.4 to 7.8, respectively. The dilution water contained only four salts with the following ionic concentrations (mg/L): Ca14, Mg 12, Na 26.3, K2, CO/" 70.6, Cl" 1.9, and SO/" 48. Four diets were tested: 1) A mixture of Yeast-CerophyllTMTrout Food (YCTF), 2) a combination of YCTF and the alga Selenastrum, 3) a feeding suspension of Selenastrum OHIO JOURNAL OF SCIENCE B. CERDA AND J. OLIVE 45 alone, and 4) a feeding concentrate of Chlamydomonas (alga). The YCTF mixture was prepared following the U.S. EPA guidelines (U.S. EPA 1989). Both algae were cultured in the algal medium described by the U.S. EPA (1989), to which a vitamin supplement (Murphy 1970) was added. Algal cultures were maintained at room temperature on a 16 h light, 8 h dark photoperiod under fluorescent and Gro-luxTM lights. Algae were allowed to grow for seven to ten days before being fed to daphnids. For feeding purposes, algae were concentrated by centrifugation. The algal density in the concentrates was determined by optical density using a spectrophotometer. To achieve the desired cell density, the concentrated algae were resuspended in the supernatants (or further concentrated) until a spectrophotometric absorbance of 1.5 at 665 nm was reached. This method of concentration provided a cell count of 3.0 to 3-5 x 10 cells/ml in the concentrates used to feed the test animals. Algal feeding suspensions and cultures were routinely inspected for viability and foreign algal contamination. Each test organism was fed 0.1 ml of the appropriate diet every day. For the combination YCTF + Selenastrum, the daily ration consisted of 0.05 ml YCTF and 0.05 ml Selenastrum. Each toxicity test consisted of ten young (<24 h old) C. dubia at each of the following copper concentrations: 0 (control), 5, 10, 20, 30, 40, 50, 60,70, 80, 90, and 100 jag/L. Each test was repeated until two valid toxicity tests (a minimum of 80% survival and 15 young/female in controls) were obtained for each of the diets evaluated. In the case of YCTF + Selenastrum, two of two tests were valid, for YCTF alone two of three were valid, and for Chlamydomonas two of five were valid. For Selenastrum alone eight tests were run, all of which met the survival requirement but failed to meet the reproduction requirement. However, two of those tests had a mean young production by the controls that was not significantly lower than 15 young/female (12.9 and 13-3), based on hypothesis testing (P <0.001). Copper was added as reagent grade CuSO4 5H2O. A stock solution of 100 mg Cu/L was prepared daily in glass distilled water. Test concentrations were obtained by diluting appropriate aliquots of the stock in the dilution water. Test concentrations were prepared daily during the