### 1 ACUTE TOXICITY TO AQUATIC INVERTEBRATES (E.G., DAPH NIA)

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 remark.)

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 850.1010 and ASTM Method E729-96 (where differences in the two method guidan ce documents exist, ASTM requirements were used) Test type: 48-hour Static-Renewal Acute GLP (Y/N): Yes Year (study performed): 2012 Analytical procedures: EPA Method 8330A was used following USEPA (1986) guidelines to analyze for nitroaromatics and nitramines by HPLC Species/Strain: Daphnia magna Test details (static, semi-static, dosing rate, flow-through rate, etc.): Static-renewal (daily) Statistical methods: Statistical analyses were n ot required for this study. If they were, test data would be used to calculate the 24- and 48-hour EC 50 values (the experimentally derived concentration of a test substance in dilution water that is calcu lated to affect 50 percent of a test population dur ing a continuous exposure over a specified period of time compared to a control population), along with their 95% confidence limits, following USEPA (2002) guidance, and based on time-weighted measured concentrations.

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 organisms  source, supplier, any pretreatment, breeding meth od: Test organisms were obtained from in-house cultures and were acclimated to test temperature fo r at least 48 hrs prior to test initiation. No deviations from method guidance.

 Age at study initiation: < 24 hours old. No devia tions or differences from method guidance.

 Control group: Two control groups were tested: a dilution water control (un-spiked dilution water) and a solvent control (0.5 ml technical grade aceto ne 

### (CAS # 67-64-1) /L dilution water). No deviations from method guidance.

- Test conditions  Stock solutions preparation (vehicle, solvent, co ncentrations) and stability: No deviations from method guidance. The test substance, PETN, was rece ived in solvent at a nominal concentration of 5,000 mg PETN/L acetone (CAS No. 78-11-5). The sto ck solution was stored at approximately 4°C in the amber jar while not in use. The stock solution was warmed to room temperature prior to use in testing.

 Test temperature range: 20 ± 2oC (actual measured temperatures ranged from 19-22oC). No deviations or differences from method guidance.

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 Exposure vessel type (e.g., size, headspace, seal ed, aeration, # per treatment): 500-ml plastic cups were used for test chambers. Test chambers were cov ered and there was headspace above solutions. Solutions were not aerated and there were four repl icates per treatment. No deviations 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, Ca/Mg ratio, Na/K ratio): No deviations from method guidance. The water was adju sted to a target hardness and alkalinity of 170 ± 10 and 115 ± 5 mg/L as CaCO 3 , respectively. Measured hardness and total alkalin ity were 172 and 116 mg/L as CaCO 3 , respectively, and the pH was 8.5 s.u. Calcium sul fate (CaSO 4 ·2H 2 O) and magnesium sulfate (MgSO 4 ) were added at a ratio of 1:1 on a mass basis to a chieve 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 a dequate organism performance (nominal concentration of 2 μg/L). TOC, TSS, salinity, and N a/K ratio were not determined.

 Lighting (quality, intensity and periodicity): 

### Lighting was controlled to provide 16 hours of ligh t and 8 hours of dark per 24-hour period; light intensity w as ambient (approximately 50 - 100 foot-candles). No deviations or differences from method guidance.

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

There was no effect observed in this study. Solvent control: D.O. ranged from 7.1 – 7.3 mg/L, p H ranged from 8.5 – 8.6 s.u. Dilution water control: D.O. ranged from 7.0 – 7.2 mg/L, pH was 8.5 s.u. All other treatments: D.O. ranged from 6.9 – 7.2 mg /L, pH ranged from 8.5 – 8.6 s.u.

- Element (unit) basis (i.e. immobilization): Immob ilization and any abnormal behavior or appearance.

- Test design (number of replicates, individuals pe r replicate, concentrations): Organisms were exposed to five concentrations of PETN (using a dil ution factor of approximately 1.5X), a solvent control (0.5 ml acetone/L dilution water), and a di lution water control. 4 replicates of each concentration were tested, with 5 organisms per rep licate. No deviations from method guidance.

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

- Exposure period: 48 hours. No deviations or diffe rences from method guidance.

- Analytical monitoring: Samples for PETN analysis were collected from freshly prepared bulk solutions at test initiation, after distributing the test sol utions to the test chambers, for each test treatmen t, the solvent control, and the dilution water. On day 1, samples were collected from freshly prepared bulk solutions of all test treatments after distributing to test chambers, as well as from all replicates o f old test solutions after organisms had been transferred to new solutions. At test termination, each replicate of the old test solutions was sampled. Al l samples were poured directly into certified clean 500-ml glass amber bottles and stored at approximat ely 4oC in the dark prior and during shipment to the analytical laboratory for analysis. No deviatio ns from method guidance.

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RESULTS

Nominal concentrations in mg/L: 0 (Solvent Control), 0 (Dilution water control), 0. 5, 0.8, 1.1, 1.7, and 2.5 mg PETN/L Measured concentrations in mg/L: <0.00076, <0.00082, 0.268, 0.419, 0.547, 0.809, 0.9 43 mg PETN/L Unit [results expressed in what unit]: mg PETN ( measured)/L EC50, EL50, LC0, LL0, at 24, 48 hours:

24-hr EC50: >0.943 mg PETN (measured)/L 48-hr EC50: >0.943 mg PETN (measured)/L Statistical results, as appropriate: Not applica ble

Remarks field for Results. Discuss if element ef fect concentration is not less than materials solubility. Describe additional information that ma y be needed to adequately assess data for reliabili ty and use including the following as appropriate:

- Biological observations: All organisms survived w ith no obvious differences in appearance between those in the control versus those in PETN treatment s.

 Number immobilized as compared to the number expo sed: 0

 Concentration response with 95% confidence limits : N/A

 Cumulative immobilization: None

 Was control response satisfactory (yes/no/unknown ): Yes

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 D. magna 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- and 48-hour EC 50 values based on the average PETN concentrations mea sured in test solutions were greater than the highest con centration tested, and there were no obvious differences in appearance between organisms in the control treatments versus those in the PETN treatments. The 95% confidence intervals could not be determined since mortality did not exceed 50% in any of the test treatments. The highest mea sured average concentration of PETN in these studies (using acetone as the solvent carrier) was 0.943 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 Cladoceran (Daphnia magna) Under Static-Renewal Test Conditions were reviewed for compliance with the test protocol and SOPs. In addition, the actual conduct of the study was reviewed for compliance. The results of “in-life” audits and ad ditional reviews were made available to the Study 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 Cladoceran (Daphnia magna) Under Static-Renewal Con ditions comply with the Final Rule for Good Laboratory Practices under the Toxic Substances Con trol Act (TSCA, 40 CFR Part 792) with the following exception: the analytical data were gener ated following NELAP (National Environmental Laboratory Accreditation Conference) guidelines. C haracterization, storage stability, and verificatio n of test substance identity are the responsibility o f 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. Aquatic Invertebrate Acute Toxicity Tes t, Freshwater Daphnids. OPPTS 850.1010. Ecological Effects Test Guidelines. EPA 712-C-96-1 14.

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.

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