### 1 ACUTE TOXICITY TO FISH

TEST SUBSTANCE

Identity: 

### 1,3-Dinitrato-2,2-bis(nitratomethyl)propane (i.e., pentaerythritol tetranitrate; CAS No. 78- 11-5)

Remarks field for Test Substance (Use for any pe rtinent, test substance-specific remarks.)

A 5,000 mg PETN/L acetone stock prepared by the sup plier was used for preparation of all test concentrations.

METHOD

Method/guideline followed (experimental/calculat ed): OPPTS Method 850.1075 and ASTM Method E 729-96 (where differences in the two method guida nce documents exist, ASTM requirements were used) Type (test type): 96-hour Static-Renewal Acute GLP (Y/N): Yes Year (study performed): 2012 Species/Strain/Supplier: Pimephales promelas (fathead minnow); in-house culture Analytical monitoring: EPA Method 8330A was used following USEPA (1986) guidelines to analyze for nitroaromatics and nitramines by HPLC Exposure period (unit): 96 hours Statistical methods: Statistical analyses were n ot required for this study. If they were, test data would be used to calculate the 24-, 48-, 72-, and 9 6-hour EC 50 values (the experimentally derived concentration of a test substance in dilution water that is calculated to affect 50 percent of a test population during a continuous exposure over a spec ified period of time compared to a control population), along with their 95% confidence limits , following USEPA (2002) guidance.

Remarks field for Test Conditions. Detail and di scuss any significant protocol deviations, and detail differences from the guideline followed incl uding the following as appropriate:

- Test fish (Age/length/weight, loading, pretreatme nt): 14 days old and <3.0 g per fish (mean weight and length of dilution water control fish at test t ermination were 0.0024 g and 9 mm, respectively; loading rate=0.096 g/L). Fish were acclimated to th e test temperature for at least 7 days prior to testing and dilution water for 5 days. Organisms we re not fed for 48 hrs prior to testing or during th e test. No deviations or differences from method guid ance.

- Test conditions, e.g.  Details of test (static, semi-static, flow-throug h): Static-renewal conditions were used. No deviati ons from method guidance.

 Dilution water source: No deviations from method guidance; the dilution/control water used in this study was 100% Milli-Q ® (ASTM type I; Millipore, Billerica, MA) water (18 megohmcm resistance water) adjusted to a target hardness and alkalinity

.

 Dilution water chemistry (hardness, alkalinity, p H, TOC, TSS, salinity): No deviations from method guidance. The water was adjusted to a target hardne ss and alkalinity of 170 ± 10 and 115 ± 5 mg/L as CaCO 3 , respectively. Measured hardness and total alkalin ity were 174 and 115 mg/L as CaCO 3 , respectively, and the pH was 8.3 s.u. Calcium sulfa te (CaSO 4 ·2H 2 O) and magnesium sulfate (MgSO 4 ) were added at a ratio of 1:1 on a mass basis to ach ieve the target hardness. Sodium bicarbonate (NaHCO 3 ) was added to increase the alkalinity. Selenium (a s Na 2 SeO 4 ) was added to this reconstituted water to maintain adequate organism performance (no minal concentration of 2 μg/L). TOC, TSS, salinity, and Na/K ratio were not determined.

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 Stock and test solution and how they are prepared : No deviations from method guidance. The test substance, PETN, was received in solvent at a nomin al concentration of 5,000 mg PETN/L acetone (CAS No. 78-11-5). The stock solution was stored a t approximately 4°C in the amber jar while not in use. The stock solution was warmed to room tempera ture prior to use in testing. All test concentrations were prepared daily prior to test so lution renewal.

 Concentrations dosing rate, flow-through rate, in what medium: The OPPTS method guidance states that a minimum of 5 concentrations must be tested i n a geometric series. However, based on preliminary studies performed by the testing labora tory (i.e., LC 50 > water solubility), it was determined that only two concentrations of PETN nee ded to be tested – 1.0 and 2.0 mg PETN/L (nominal).

 Vehicle/solvent and concentrations: Acetone was u sed as a solvent carrier. The highest concentration tested was 0.4 ml acetone technical grade/L. No dev iations from method guidance.

 Stability of the test chemical solutions: Assumed to be stable in solution.

 Exposure vessel type (e.g., size, headspace, seal ed, aeration, lighting, # per treatment): 500-ml plastic cups were used for test chambers. Test cham bers were covered and there was headspace above solutions. Test solutions were not aerated an d there were three replicates per treatment. No deviations from the method guidance.

 Number of replicates, fish per replicate: Three r eplicates were tested for each treatment, each with 10 organisms (total of 30 organisms per treatment). No deviations from method guidance.

 Water chemistry in test (D.O., pH) in the control and one concentration where effects were observed:

Solvent control: D.O. ranged from 6.4 – 6.9 mg/L, p H ranged from 8.2 – 8.4 s.u. Dilution water control: D.O. ranged from 6.3 – 6.8 mg/L, pH ranged from 8.3 – 8.4 s.u. 1,000 and 2,000 μg PETN/L (nominal): D.O. ranged fr om 6.2 – 6.8 mg/L, pH ranged from 8.3 – 8.4 s.u.

- Test temperature range: 25 ± 2oC (actual measurem ents ranged from 23.0 to 25.1oC). No deviations or differences from method guidance.

- Method of calculating mean measured concentration s (i.e. arithmetic mean, geometric mean, etc.): Arithmetic mean. No deviations or differences from method guidance.

RESULTS

Nominal concentrations (as mg/L): 0 (Solvent con trol), 0 (Dilution water control), 1.0, and 2.0 mg PETN/L Measured concentrations (as mg/L): <0.00072, <0. 00070, 0.723, and 1.218 mg PETN/L Unit (results expressed in what unit): mg PETN ( measured)/L Element value (e.g. LC 50 , LC o , LL 50 , or LL 0 at 48, 72 and 96 hours, etc., based on measured or nominal concentrations)

24-hr EC50: >1.218 mg PETN (measured)/L 48-hr EC50: >1.218 mg PETN (measured)/L 72-hr EC50: >1.218 mg PETN (measured)/L 96-hr EC50: >1.218 mg PETN (measured)/L

Statistical results, as appropriate: N/A

### 3 Remarks field for Results. Discuss if element ef fect concentration is greater than materials solubility. Describe additional information that ma y be needed to adequately assess data for reliabili ty and use, including the following, if available:

- Biological observations: Organisms appeared to be weaker in both of the PETN concentrations than those in the control treatments. - Table showing cumulative mortality



### Nominal Treatment (mg PETN/L) % Mortality at 96 hours

Rep A Rep B Rep C Overall Mortality Std.

Dev. 0 (Solv. Cont) 

### 0 10 0 3.3 5.77 0 (Dil. Cont.) 

### 0 0 0 0 0.00

1.0 

### 0 10 0 3.3 5.77
2.0 

### 30 20 30 26.7 5.77

- Lowest test substance concentration causing 100% mortality: >1.218 mg PETN/L (measured) - Mortality of controls: Solvent control: 3.3% / Di lution water control: 0% - Abnormal responses: None - Reference substances (if used)- results: None - Any observations, such as precipitation that migh t cause a difference between measured and nominal values: Due to the low solubility of PETN i n water, small amounts of PETN crystals could be seen precipitating out of solution and sticking to the sides of glassware. Care was taken to keep as many of these crystals as possible in the test solu tion. The low solubility can account for the differ ence between the nominal and measured concentrations.

CONCLUSIONS

Remarks field with the ability to identify sourc e of comment, i.e. author and/or submitter

The purpose of this study was to determine the acut e toxicity of PETN to P. promelas under staticrenewal test conditions. Due to problems getting t he PETN in solution to its determined water solubility (~2.0 mg/L in ultrapure water) because o f the salt content (polarity) of the dilution water
, this study was performed using a solvent carrier. The biological endpoint for this study was mortalit y (or survival). The 24-, 48-, 72-, and 96-hour EC 50 values based on the average PETN concentrations measured in test solutions for the definitive acute P. promelas study were all greater than the highest concentration tested. The 95% confidence intervals could not be determined since mortality did not exceed 50% in any of the test treatments. The high est measured average concentration of PETN in these studies (using acetone as the solvent carrier ) was 1.218 mg PETN/L, lower than the reported water solubility of PETN which is ~2.0 mg/L.

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DATA QUALITY

Reliabilities (Klimisch Code, if used, possibly a flag if 'key study')

Remarks field for Data Reliability

The raw toxicity data and report for the study, Acute Toxicity of Pentaerythritol tetranitrate (PET N) to the Fathead Minnow (Pimephales promelas) Under Static-Renewal Test Conditions , were reviewed for compliance with the test protocol and SOPs. In add ition, the actual conduct of the study was reviewed for compliance. The results of “in-life” audits an d additional reviews were made available to the Stu dy Director for corrective action. The QA statement do es not include the analytical data; though the data were determined to be accurate and useable.

The biological data and report presented for Acute Toxicity of Pentaerythritol tetranitrate (PET N) to the Fathead Minnow (Pimephales promelas) Under Static-Renewal Test Conditions , comply with the Final Rule for Good Laboratory Practices under the Toxic Substances Control Act (TSCA, 40 CFR Part 792) with the following exception: the analytical data w ere generated following NELAP (National Environmental Laboratory Accreditation Conference) guidelines. Characterization, storage stability, and verification of test substance identity are the responsibility of the Study Sponsor and are not encompassed in this compliance statement.

REFERENCES (Free Text)

ASTM. 2009. Standard Guide for Conducting Acute T oxicity Tests on Test Materials with Fishes,

Macroinvertebrates, and Amphibians. Method E 729-9 6 in 2009 Annual Book of ASTM Standards, Section 11, Water and Environmental Tech nology, Volume 11.06, Biological Effects and Environmental Fate; Biotechnology. American So ciety of Testing and Materials.

OPPTS 1996. Fish Acute Toxicity Test, Freshwater an d Marine. OPPTS 850.1075. Ecological Effects Test Guidelines. EPA 712-C-96-118.

USEPA. 1986. Test Methods for Evaluating Solid Wast e, Physical/Chemical Methods. Nitroaromatics and Nitramines 8330: Third Edition. SW-846.

USEPA. 1989. Good Laboratory Practice Standards. Final Rule. 40 CFR Part 792 (from 54 FR 34043, August 17, 1989).

USEPA. 2002. Methods for Measuring the Acute Toxi city of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fifth Edition. EP A-821-R-02-012.

OTHER

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