Toxicity Report. Section 1. Overview

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1 Toxicity Report Date: October 25, 2010 JSC SPACE TOXICOLOGY GROUP Noreen N. Khan-Mayberry, Ph.D. NASA Space Toxicologist Johnson Space Center, SF 23 Houston, TX Ref. Acute Aquatic Toxicity- AKGA Voice: Fax: BB: Section 1. Overview The Space Toxicology office was asked to provide a toxicity assessment of 3 candidate chemicals AKGA (alpha-ketoglutaric acid), PCA (3-Pyridazinecarboxylic acid, 1,4,5,6-tetrahydro- 1-methyl-6-oxo-), and MPCA (1-Methyl-6-oxo-1,4,5,6-tetrahydropyridazine-3-carboxylic acid). These chemicals are propellant reactants that will be used to decrease toxicity of hydrazine (HZ) and/or monomethylhydrazine (MMH). The subsequent byproducts will be disposed of as treated effluent (wastewater). Acute toxicity testing was conducted on AKGA, AKGA +HZ and AKGA+MMH. The results from these tests are the focus of this report. The acute toxicity of PCA and MPCA is not available, since these samples have not been tested for toxicity. A static renewal test was recommended since Static tests provide the most easily obtained measure of acute toxicity. Results of acute toxicity tests should usually be reported in terms of a median lethal concentration (LC50) or median effective concentration (EC50). In some situations, it might be necessary only to determine whether a specific concentration is acutely toxic to the test species or whether the LC50 or EC50 is above or below a specific concentration (ASTM E ). Renewal tests refer to an assay where the test toxicant is replaced (or renewed) at defined intervals while the test species is maintained throughout the evaluation period. Four species of test species (2 feshwater and 2 saltwater) were used in the bioassays. The four test species used in the bioassays are: 1. Ceriodaphnia dubia ( common name: water flea freshwater species), 2. Cyprinella leedsi (common name: Bannerfin Shiner freshwater species), 3. Mysidopsis bahia (common name: Mysid Shrimp saltwater species), 4. Menidia beryllina (common name: Silverside Minnow saltwater species) The method used for all bioassay tests in this document is: EPA-832-R : U.S. Environmental Protection Agency (EPA). Methods for Measuring the Acute Toxicity of Effluents and Receiving Water to Freshwater and Marine Organisms. (2002, 5 th Edition).

2 Section 2. Results Acute toxicity tests conducted by independent laboratory Hydrosphere Research laboratory specifically for NASA s use through a Wyle Science & Engineering Group subcontract. See Appendix A for full report from Hydrosphere.

3 Section 3. Discussion All 3 samples, AKGA, AKGA + HZ and AKGA + MMZ were tested for acute toxicity and show relatively low toxicity when diluted to at least 0.001M. These samples will henceforth be referred to as samples A, B & C. Sample A (AKGA): 1M AKGA diluted to 0.002M with H 2 0 and adjusted to a ph of 8 with NaOH. Sample B (AKGA + HZ) 1M AKGA + HZ diluted to 0.002M with H 2 0 and adjusted to a ph of 8 with NaOH. Sample C (AKGA + MMZ) 1M AKGA + MMZ diluted to 0.002M with H 2 0 and adjusted to a ph of 8 with NaOH. Freshwater Bioassay C. dubia (Water flea) The results of this assay showed the highest level of toxicity with LC 50 survival rates of 66% for Sample A; 71% for Sample B and & 71% for sample C. Sample A 0% survival rate at zero dilution or 100% strength of original delivered sample. 95% survival rate at 50% strength of original delivered sample. 95% survival rate at 12.5% strength of original delivered sample. Sample A is highly toxic to C.dubia at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). This concentration is NOT RECOMMENDED for effluent disposal due to its high acute toxicity in the C.dubia species. Sample A shows very low acute toxicity to zero acute toxicity when diluted to at least 50% of its original concentration strength (0.001M). Sample A is safe for effluent disposal when diluted to at least 50% of the original sample concentration based upon acute toxicity results in C.dubia. Sample B 0% survival rate at zero dilution or 100% strength of original delivered sample.

4 Sample B is highly toxic to C.dubia at 0.002M concentration (1M AKGA + HZ diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). This concentration is NOT RECOMMENDED for effluent disposal due to its high acute toxicity in the C.dubia species. Sample B demonstrates zero acute toxicity when diluted to at least 50% of its original concentration strength(0.001m). Sample B is safe for effluent disposal when diluted to at least 50% of the original sample concentration based upon acute toxicity results in C.dubia. Sample C 0% survival rate at zero dilution or 100% strength of original delivered sample. Sample C is highly toxic to C.dubia at 0.002M concentration (1M AKGA + MMZ diluted to 0.002M with H 2 0 and adjusted to a ph of 8 with NaOH). This concentration is NOT RECOMMENDED for effluent disposal due to its high acute toxicity in the C.dubia species. Sample C demonstrates zero acute toxicity when diluted to at least 50% of its original concentration strength (0.001M). Sample C is safe for effluent disposal when diluted to at least 50% of the original sample concentration based upon acute toxicity results in C.dubia. Freshwater Bioassay C. leedsi (Bannerfin Shiner) The results of this assay showed LC 50 survival rates of >100% for Sample A; >100% for Sample B and & >100% for sample C. Sample A 90% survival rate at zero dilution or 100% strength of original delivered sample. Sample A is shows very low acute toxicity to zero acute toxicity to C. leedsi at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). Although this sample shows low toxicity in C.leedsi, this concentration is NOT RECOMMENDED for effluent disposal due to its high acute toxicity in the C.dubia species.

5 Sample A shows very low acute toxicity to zero acute toxicity when diluted to at least 50% of its original concentration strength (0.001M). Sample A is safe for effluent disposal when diluted to at least 50% of the original sample concentration based upon acute toxicity results in C. leedsi. Sample B 100% survival rate at zero dilution or 100% strength of original delivered sample. Sample B shows zero acute toxicity to C. leedsi at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). Although this sample shows no toxicity in C.leedsi, this concentration is NOT RECOMMENDED for effluent disposal due to its high acute toxicity in the C.dubia species. Sample B demonstrates zero acute toxicity when diluted to at least 50% of its original concentration strength(0.001m). Sample B is safe for effluent disposal when diluted to at least 50% of the original sample concentration based upon acute toxicity results in C. leedsi. Sample C 100% survival rate at zero dilution or 100% strength of original delivered sample. Sample C shows zero acute toxicity to C. leedsi at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). Although this sample shows no toxicity in C.leedsi, this concentration is NOT RECOMMENDED for effluent disposal due to its high acute toxicity in the C.dubia species. Sample C demonstrates zero acute toxicity when diluted to at least 50% of its original concentration strength (0.001M). Sample C is safe for effluent disposal when diluted to at least 50% of the original sample concentration based upon acute toxicity results in C. leedsi. Salwater Bioassay Mysidopsi bahia (Mysid Shrimp) The results of this assay showed LC 50 survival rates of >100% for Sample A; >100% for Sample B and & >100% for sample C.

6 Sample A 90% survival rate at zero dilution or 100% strength of original delivered sample. 65% survival rate at 50% strength of original delivered sample. 90% survival rate at 25% strength of original delivered sample. 90% survival rate at 12.5% strength of original delivered sample. 95% survival rate at 6.25% strength of original delivered sample. Sample A is shows very low acute toxicity to zero acute toxicity to M. bahia at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). Although this sample shows low toxicity in M. bahia, this concentration is NOT RECOMMENDED for effluent disposal due to its high acute toxicity in the C.dubia species. Sample A demonstrated the highest level of toxicity at 50% strength (0.001M) showing a 65% survival rate. While these results do not reflect why lower toxicity was seen at full strength, they are still considered in the overall recommendation. Sample A shows very low acute toxicity to zero acute toxicity when diluted to at least 25% of its original concentration strength (0.0005M). Sample A is safe for effluent disposal at all dilution strengths tested in M. Bahia. Sample B 80% survival rate at zero dilution or 100% strength of original delivered sample. 90% survival rate at 50% strength of original delivered sample. 70% survival rate at 12.5% strength of original delivered sample. 85% survival rate at 6.25% strength of original delivered sample. Sample B shows a range of toxicity that does not correspond with molar strength; the survival rate of 80% (zero dilution M) as the dilution ratio increases to 25% (0.0005M) which showed 100% survival (no toxicity). However, the highest level of toxicity is seen at the next level of dilution of 12.5% strength ( M) showing a 70% survival rate. 85% survival is seen at the highest dilution 6.25% (0.0001M). Overall sample B shows mild toxicity to M. bahia. The weight of evidence in M. bahia and results in the freshwater C. leedsi demonstrate that Sample B is safe for effluent disposal when diluted to at least 25% of the original sample concentration. Sample C 90% survival rate at zero dilution or 100% strength of original delivered sample.

7 95% survival rate at 50% strength of original delivered sample. 85% survival rate at 25% strength of original delivered sample. 95% survival rate at 12.5% strength of original delivered sample. 85% survival rate at 6.25% strength of original delivered sample. Sample C shows low toxicity to M. bahia at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). Sample C showed highest toxicity at 25% (0.0005M) & 6.25% (0.0001M) showing a 85% survival rate; this is still considered low toxicity. Sample C is safe for effluent disposal at all dilution strengths tested in M. Bahia. Salwater Bioassay Menidia beryllina (Silverside Minnow) The results of this assay showed LC 50 survival rates of >100% for Sample A; >100% for Sample B and & >100% for sample C. Sample A 95% survival rate at zero dilution or 100% strength of original delivered sample. Sample A is shows very low acute toxicity to M. beryllina at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). Sample A demonstrates zero acute toxicity when diluted to at least 50% of its original concentration strength (0.001M). Sample A is safe for effluent disposal at all dilution strengths tested in M. beryllina. Sample B 95% survival rate at zero dilution or 100% strength of original delivered sample. Sample B shows low acute toxicity to M. beryllina at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH).

8 Sample B demonstrates zero acute toxicity when diluted to at least 50% of its original concentration strength (0.001M). Sample B is safe for effluent disposal all dilution strengths tested in M. beryllina. Sample C 100% survival rate at zero dilution or 100% strength of original delivered sample. 95% survival rate at 25% strength of original delivered sample. 95% survival rate at 6.25% strength of original delivered sample. Sample C shows zero acute toxicity to M. beryllina at 0.002M concentration (1M AKGA diluted to 0.002M concentration with H 2 0 and adjusted to a ph of 8 with NaOH). Sample C shows very low to no acute toxicity when diluted to at least 50% of its original concentration strength (0.001M). Sample C is safe for effluent disposal at all dilution strengths tested in M. beryllina. Section 5. Conclusion It is recommended that all solutions (A, B & C) be diluted to 0.001M concentration, or 50% of what was delivered at full strength for effluent disposal. This is based upon zero survival in the C.dubia species (water flea) when exposed to all samples at 0.002M concentration. All other dilutions show mild to no acute toxicity in all species tested. Raw test data is included in Hydrosphere report in Appendix A. It is further recommended that chronic toxicity testing (7 day chronic static renewal) to determine the long term toxic effects. We appreciate the opportunity to assist Kennedy Space Center in determining acute toxicity of candidate chemicals. If you have any further questions regarding this report and recommendations, please contact us. Prepared & signed by: Noreen Khan-Mayberry, Ph.D. NASA Space Toxicologist

9 APPENDIX A HYDROSPHERE LABORATORIES 96 HOUR ACUTE STATIC RENEWAL TESTING RESULTS