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ACUTE TOXICITY TO AQUATIC INVERTEBRATES (E.G., DAPHNIA) 

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 pertinent, test substance-specific remark.) 

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

concentrations. 

METHOD

 Method/guideline followed (experimental/calculated): OPPTS Method 850.1010 and ASTM Method 

E729-96 (where differences in the two method guidance 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 not required for this study. If they were, test data

would be used to calculate the 24- and 48-hour EC50 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 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 discuss any significant protocol deviations and 

detail differences from the guideline followed including the following as appropriate: 

- Test organisms 

 source, supplier, any pretreatment, breeding method: Test organisms were obtained from in-house 

cultures and were acclimated to test temperature for at least 48 hrs prior to test initiation. No 

deviations from method guidance. 

 Age at study initiation: < 24 hours old. No deviations 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 acetone (CAS # 67-64-1)/L dilution water). No deviations 

from method guidance. 

- Test conditions 

 Stock solutions preparation (vehicle, solvent, concentrations) and stability: No deviations from 

method guidance. The test substance, PETN, was received in solvent at a nominal concentration of 

5,000 mg PETN/L acetone (CAS No. 78-11-5). The stock 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, sealed, aeration, # per treatment): 500-ml plastic cups

were used for test chambers. Test chambers were covered and there was headspace above solutions. 

Solutions were not aerated and there were four replicates 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, pH, TOC, TSS, salinity, Ca/Mg ratio, Na/K ratio): No

deviations from method guidance. The water was adjusted to a target hardness and alkalinity of 170 ± 

10 and 115 ± 5 mg/L as CaCO3, respectively. Measured hardness and total alkalinity were 172 and 

116 mg/L as CaCO3, respectively, and the pH was 8.5 s.u. Calcium sulfate (CaSO4·2H2O) and 

magnesium sulfate (MgSO4) were added at a ratio of 1:1 on a mass basis to achieve the target 

hardness. Sodium bicarbonate (NaHCO3) was added to increase the alkalinity. Selenium (as Na2SeO4) 

was added to this reconstituted water to maintain adequate organism performance (nominal 

concentration of 2 μg/L). TOC, TSS, salinity, and Na/K ratio were not determined. 

 Lighting (quality, intensity and periodicity): Lighting was controlled to provide 16 hours of light and 8 

hours of dark per 24-hour period; light intensity was 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, pH 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): Immobilization and any abnormal behavior or appearance. 

- Test design (number of replicates, individuals per replicate, concentrations): Organisms were 

exposed to five concentrations of PETN (using a dilution factor of approximately 1.5X), a solvent 

control (0.5 ml acetone/L dilution water), and a dilution water control. 4 replicates of each 

concentration were tested, with 5 organisms per replicate. No deviations from method guidance. 

- Method of calculating mean measured concentrations (i.e. arithmetic mean, geometric mean, etc.): 

Arithmetic mean (equivalent to time-weighted average). No deviations or differences from method 

guidance. 

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

- Analytical monitoring: Samples for PETN analysis were collected from freshly prepared bulk solutions

at test initiation, after distributing the test solutions to the test chambers, for each test treatment, 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 of old 

test solutions after organisms had been transferred to new solutions. At test termination, each 

replicate of the old test solutions was sampled. All samples were poured directly into certified clean

500-ml glass amber bottles and stored at approximately 4oC in the dark prior and during shipment to 

the analytical laboratory for analysis. No deviations 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, 0.943 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 applicable 

 Remarks field for Results. Discuss if element effect concentration is not less than materials 

solubility. Describe additional information that may be needed to adequately assess data for reliability 

and use including the following as appropriate: 

- Biological observations: All organisms survived with no obvious differences in appearance between 

those in the control versus those in PETN treatments. 

 Number immobilized as compared to the number exposed: 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 source of comment, i.e. author and/or submitter: 

The purpose of this study was to determine the acute toxicity of PETN to D. magna under staticrenewal test conditions. Due to problems getting the PETN in solution to its determined water 

solubility (~2.0 mg/L in ultrapure water) because of the salt content (polarity) of the dilution water, 

this study was performed using a solvent carrier. The biological endpoint for this study was mortality 

(or survival). The 24- and 48-hour EC50 values based on the average PETN concentrations measured 

in test solutions were greater than the highest concentration 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 measured 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 (PETN) 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 additional reviews were made available to the Study 

Director for corrective action. The QA statement does 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 (PETN) to the 

Cladoceran (Daphnia magna) Under Static-Renewal 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 were 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 Toxicity Tests on Test Materials with Fishes, 

Macroinvertebrates, and Amphibians. Method E 729-96 in 2009 Annual Book of ASTM 

Standards, Section 11, Water and Environmental Technology, Volume 11.06, Biological Effects 

and Environmental Fate; Biotechnology. American Society of Testing and Materials. 

OPPTS 1996. Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids. OPPTS 850.1010. 

Ecological Effects Test Guidelines. EPA 712-C-96-114. 

USEPA. 1986. Test Methods for Evaluating Solid Waste, 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 Toxicity of Effluents and Receiving Waters to 

Freshwater and Marine Organisms. Fifth Edition. EPA-821-R-02-012. 

OTHER

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