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RECYCLEO \ -Ja> \ O-fa. svf'wsw 89=8* n" TRADE SECRET ARiW -i S DuPont-11409 Study Title H-25434: Static, Acute, 48-Hour Screening Test to Daphnia magna Laboratory Project ED: DuPont-11409 Author: Laurie D. Bouchelle Alan Samel, M.S. Study Completed on: October 2,2002 Performing Laboratory: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request Number: Study Code Number: Page 1 o f 7 Company Sanitized. Does not contain TSCA CBI H-25434: Static, Acute, 48-Hour Screening Test to Daphnia magna DuPont-11409 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study Director: _ Associate Scientist Date -2 Company Sanitized. Does not contain TSCA C8I H-25434: Static, Acute, 48-Hour Screening Test to Daphnia magna STUDY INFORMATION Substance Tested: Svnonvms/Codes: H-25434 Haskell Number: 25434 Composition:] DuPont-11409 Known Impurities: Not characterized Physical Characteristics: Off-white, milky semi-opaque aqueous dispersion Stability: The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: September 24,2002 / (see report cover page) In-Life Initiated/Completed: September 24, 2002 / September 26, 2002 -3 Company Sanitized. Does not contain TSCA CBi H-25434: Static, Acute, 48-Hour Screening Test to Daphnia magna DuPont-11409 SUMMARY The acute toxicity o f H-25434 to the water flea, Daphnia magna (less than 24 hours old) was determined in an unaerated, 48-hour, static test. The study was conducted with 4 concentrations of H-25434 and a dilution water control at a mean temperature of 20.6C (range of 20.5-20.7C). One test chamber was used per test concentration with 10 test organisms in each chamber. Based on visual observations, the water control and the 1.0, 10 and 100 mg/L test concentrations were clear with no color at test start. The 1000 mg/L test concentration was cloudy at test start. All water quality parameters were within acceptable limits during the exposure. Exposure of water fleas to the water control and nominal H-25434 concentrations o f 1, 10, 100, and 1000 mg/L resulted in 0, 0, 0,10, and 100% immobility, respectively, at the end o f 48 hours. No immobility or sublethal effects were seen in the water control test organisms. The highest nominal concentration causing no immobility at test end was 10 mg/L. The lowest nominal concentration causing 100% immobility at test end was 1000 mg/L. Nominal H-25434 concentrations were used for calculation o f EC50 values. The results are summarized as follows: Nominal concentrations o f H-25434, mg/L 48-hour EC50 (95% fiducial limits) for H-25434, based on nominal concentrations, mg/La dilution water control, 1, 10, 100, and 1000 234 (85 to 592) H-25434 exhibited low concern for aquatic hazardb in an unaerated, 48-hour, static, acute test using the water flea, Daphnia magna (less than 24 hours old). Peltier, W.H. and Weber, C.I., Eds. (1985). Methodsfo r Measuring the Acute Toxicity o f Effluents to Freshwater and Marine Organisms. EPA/600/4-85-013. United States Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio. b Smrchek, J., Clements, R., Morcock, R., and Rabert, W. (1993). A ssessing ecological hazard under TSCA: methods and evaluation o f data. Environmental Toxicology and Risk Assessment, ASTM STP 1179. (W .G. Landis, J.S. Hughes, and M.A. Lewis, Eds.), pp 22-39. American Society for Testing and Materials, Philadelphia. -4 Company Sanitized. Does not contain TSCA CBI H-25434: Static, Acute, 48-Hour Screening Test to Daphnia magna DuPont-11409 MATERIALS AND METHODS A. Test Solution Preparation Test substance solutions were prepared by direct addition of H-25434 to dilution water. Test solutions o f 1, 10,100, and 1000 mg/L were prepared by adding the appropriate amount o f test material to HLWW in 1-L beakers and stirring for approximately 30 minutes. The water control and the 1.0, 10 and 100 mg/L test concentrations were clear with no color at test start. The 1000 mg/L test concentration was cloudy at test start. B. Dilution Water Dilution water originated from the Haskell Laboratory well which is 378-feet deep and is cased and sealed to bedrock. The hardness o f the HLWW is adjusted to approximately 100-140 mg/L as CaCC>3 by the flow-proportioned addition of CaC^. The HLWW is then aerated, passed through a green sand filter to remove iron, and filtered through 50-, 10-, and 5-pm filters to remove particulates. The water is heated or chilled as appropriate and distributed through aged polyvinyl chloride piping. The dilution water is analyzed twice yearly for major anions and cations, metals, total organochlorine and organophosphate pesticides, and polychlorinated biphenyls. The dilution water meets OECD(1) and ASTM(2) specifications. C. Test Organism Culture Daphnia magna were reared at Haskell Laboratory in 250-mL (1 per beaker) or 1000-mL Pyrex beakers (10 per beaker at culture initiation) which contained 200 mL or 1000 mL o f aerated, filtered HLWW held at 20.9C. Daphnids were fed on a daily basis with a yeast, cereal leaves and trout chow (YCT) mixture (standardized to 1700 to 2100 mg/L total solids) and the green alga, Selenastrum capricornutum, at a rate o f 62,500 cells/mL (the combination of YCT and alga is equivalent to 0.1-0.2 mg total organic carbon per daphnid). Neonates used in this test were less than 24 hours old and were collected from the 12th brood o f 28-day old parent daphnids. Sickness, injury, and abnormalities were not seen and ephippia were not being produced by the parent daphnids. No adult immobility was seen in the cultures used for testing during the 48-hour pretest period. Daphnia magna were identified by labels on the culture beakers and test chambers. D. Test Methods Four nominal concentrations and a dilution water control were used in this study. The nominal concentrations were 1, 10, 100, and 1000 mg/L H-25434. -5 Company Sanitized. Does not contain TSCA CBI H-25434: Static, Acute, 48-Hour Screening Test to Daphnia magna DuPont-11409 Pyrex beakers (250-mL) containing 200 mL o f test solution (6.8-cm test solution depth) were used as test chambers. One replicate test chamber was used per test concentration with 10 daphnids in each chamber. The test chambers were covered with a glass plate during the test. Daphnia magna neonates, less than 24 hours old, were used in this study. Daphnids were not fed during the test. Addition of daphnids to test solutions was initiated after mixing of the test solutions was completed. Immobility and behavioral observations were made daily. The criterion for the effect (immobility) was a lack o f reaction to application o f a gentle stimulus. A recirculating water bath was used to maintain mean temperature in the test chambers during the 48-hour test at approximately 20.6C with a range o f 20.5 to 20.7C. A photoperiod o f 16 horns light (approximately 628 to 807 Lux) and 8 hours darkness was employed which included 30 minutes of transitional light (32 to 53 Lux) preceding and following the 16-hour light interval. Dissolved oxygen concentration, pH, and temperature were measured in all replicates of the control and test substance concentrations. These measurements were taken before daphnids were added at test start, and at test end or at total immobility in a replicate chamber. Test solutions were not aerated during the test and were disposed o f in an appropriate manner at test end. REFERENCES 1. Organisation for Economic Co-Operation and Development (OECD). Guideline for Testing Chemicals: 203, 17 July 1992.2 2. American Society for Testing and Materials (ASTM). (1988). Standard Guide for Conducting Acute Toxicity Tests with Fishes, Macroinvertebrates, and Amphibians. E 729-88a. Annual Book o f ASTM Standards, Vol. 11.04. -6 Company Sanitized. Does not contain TSCA C8I H-25434: Static, Acute, 48-Hour Screening Test to Daphnia magna DuPont-11409 TABLE 1 DISSOLVED OXYGEN AND pH OF TEST AND CONTROL SOLUTION SAMPLES Nominal H-25434 Concentration (mg/L) Test Initiation (0 hours) Dissolved Oxygen (mg/L) pH Test Completion (48 hours) Dissolved Oxygen (mg/L) pH Water Control 8.5 8.0 8.7 8.0 1.0 8.6 7.9 8.7 8.0 10 8.6 7.7 8.7 8.0 100 8.6 7.7 8.7 8.0 1000 8.6 7.7 8.8a 7.9a a Measurement performed at 24 hours due to total immobility. TABLE 2 IMMOBILITY OF Daphnia magna IN AN UNAERATED, STATIC, ACUTE, 48-HOUR TEST WITH H-25434 Nominal H-25434 Concentration (mg/L) Water Control 1.0 10 100 1000 Number o f Dead Test Organisms / Number of Test Organisms at Study Start 24 Hours 48 Hours 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 1/10 10/10 -7 Company Sanitized. Does not contain TSCA CBI