Toxicity of flame–retardants on Daphnia magna. Namuun Bayaraa, Leona Scanlan, Chris Vulpe Department of Nutritional Sciences and Toxicology; University of California, Berkeley Why Daphnia magna? Freshwater crustacean Short life span Easy to culture They mature early Parthenogenesis reproduction Figure 1: Adult D. magna with embryos in her brood chamber. ToxicantAcute LC 50 Octa-PBDE3.963mg/L Penta-PBDE58.44ug/L Firemaster550®193.7mg/L Firemaster BZ mg/L Tri-phenyl phosphate0.935 mg/L Table1. Comparison of flame-retardant acute LC 50 s. Figure 3. Acute LC 50 Toxicity Assay Figure 5. Gel electrophoresis of the RNA sample, mRNA band gap Figure 6. One color microarray, gene particles Figure 4. Chronic Toxicity Assay Flame-Retardant components Introduction Polybrominated diphenyl ethers (PBDE) are organobromine compounds that are widely used as flame-retardants. PBDEs can be found in some textiles, electronics, plastics, and household furniture such as sofas, chairs, and mattresses. These toxicants are released in indoor environments via volatilization or as dust particles, meaning the air might be polluted with them. Although use of flame-retardants might save lives and property, there have been unintended consequences. There is growing evidence that PBDEs persist in the environment and accumulate in living organisms, as well as toxicological testing that indicates these chemicals may cause liver toxicity, thyroid toxicity, and neurodevelopmental toxicity. Three major PBDE commercial mixtures have been commonly used in consumer products: deca-BDE, octa-BDE, and penta-BDE. The penta-BDE and octa-BDE commercial PBDE mixtures are no longer being produced or sold in the United States, whereas deca-BDE continues to be manufactured and sold in the United States as well as worldwide. Currently, 11 states, including California, have banned the use of penta-BDE and octa- BDE; however, the ubiquity of these chemicals combined with the slow replacement time for products previously manufactured with penta- and octa-BDE suggests that a long- term, substantial exposure reservoir will remain for some time despite PBDE phase-outs. In our project we will examine how octa-PBDE and Firemaster550® (which is a replacement for penta-PBDE) affect the eco-indicator animal Daphnia magna. Results Octa-PBDE acute LC 50 was found. Compared to Firemaster550® it was less toxic. But relevant high concentrations significantly toxic to Daphnia magna. Discussion Due to lack of time we haven’t gathered much data for gene expression analysis. We found out that some samples did not have enough RNA, so our microarray turned out not as great. In the future we will conduct octa-PBDE chronic tests, and more gene expression tests for more detailed analysis of the effect. And we will repeat the mass accumulation studies to verify our previous results. References Schecter A, Päpke O, Harris T, et al. Polybrominated Diphenyl Ether (PBDE) Levels in an Expanded Market Basket Survey of U.S. Food and Estimated PBDE Dietary Intake by Age and Sex. Environmental Health Perspectives. October 2006;114(10): U.S. Environmental Protection Agency.Polybrominated diphenylethers (PBDEs) Zota A, Rudel R, Morello-Frosch R, Brody J. Elevated House Dust and Serum Concentrations of PBDEs in California: Unintended Consequences of Furniture Flammability Standards?. Environmental Science & Technology. November 2008;42(21): Methods Octa-PBDE Acute LC 50 is determined by doing an 48 hour acute toxicity assay. 5 neonates per replicate are exposed in different concentrations and mortality rate is found by analyzing data in Probit statistical program. Using 1/10 LC 50 adult daphnids are exposed to an 48 hour assay for gene expression analysis. RNA extraction - exposed samples and control are homogenized and RNA is isolated. We measure the light absorbance a spectrophotometer and run a gel to find out if we have enough RNA and if there is any contamination. Microarray gene expression analysis – samples are analyzed using GenePix program on microarray slide. Further analysis will tell us if the genes are up or down regulated Firemaster 550® Chronic toxicity assay – 21-day lifetime toxicity assay. Number of offspring is analyzed Mass Accumulation study – mass number of adults are exposed in high chemical concentration for 48-hour, and neonates for 21-day. Acknowledgements I would like to thank Chris Vulpe, Leona Scanlan for having me in the lab and all the other members for helping me. A special thanks to Sang Lee, Lynn Huntsinger, my ELP fellows and my family for all the guidance, support and encouragement. Abstract: The flame-retarding chemicals, polybrominated diphenyl ethers, or PBDEs, are widely found in furniture cushions, carpet pads, electronics and other common household items. Even though they are used to reduce fire related injuries, they are contaminating the environment. Research on lab animals has suggested that many PBDEs mimic the effect of thyroid hormones, meddling with reproduction and nerve and tissue development. But there is not much known about how toxic flame-retardants are to eco-indicator organism Daphnia magna. The objective of the project is to determine whether these chemicals affect D.magna. We will conduct acute and chronic toxicity tests to find how and at what level flame-retardants affect D.magna. The results show that different chemicals are toxic at different concentrations. In the future we will do more chronic exposures and perform gene expression assays to further analyze effects. 6/96/236/256/286/307/27/57/7 # of neonates Table2. Mass Accumulation study death toll Figure 2. Firemaster 550® components (A-D) and octa-PBDE structure (E). (A) Isopropylated triphenyl phosphate (B) triphenyl phosphate (C) ethylhexyl 2,3,4,5-tetrabromo benzoate (D) tetrabrominated phthalate. ABCD E