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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 pertinent, test substance-specific remarks.) 

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 850.1075 and ASTM Method 

E 729-96 (where differences in the two method guidance 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 not required for this study. If they were, test data

would be used to calculate the 24-, 48-, 72-, and 96-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. 

 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 fish (Age/length/weight, loading, pretreatment): 14 days old and <3.0 g per fish (mean weight 

and length of dilution water control fish at test termination were 0.0024 g and 9 mm, respectively; 

loading rate=0.096 g/L). Fish were acclimated to the test temperature for at least 7 days prior to 

testing and dilution water for 5 days. Organisms were not fed for 48 hrs prior to testing or during the 

test. No deviations or differences from method guidance. 

- Test conditions, e.g. 

 Details of test (static, semi-static, flow-through): Static-renewal conditions were used. 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): 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 174 and 115 mg/L as CaCO3, 

respectively, and the pH was 8.3 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. 
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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 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. All test 

concentrations were prepared daily prior to test solution renewal. 

 Concentrations dosing rate, flow-through rate, in what medium: The OPPTS method guidance states 

that a minimum of 5 concentrations must be tested in a geometric series. However, based on 

preliminary studies performed by the testing laboratory (i.e., LC50 > water solubility), it was 

determined that only two concentrations of PETN needed to be tested – 1.0 and 2.0 mg PETN/L 

(nominal). 

 Vehicle/solvent and concentrations: Acetone was used as a solvent carrier. The highest concentration

tested was 0.4 ml acetone technical grade/L. No deviations from method guidance. 

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

 Exposure vessel type (e.g., size, headspace, sealed, aeration, lighting, # per treatment): 500-ml 

plastic cups were used for test chambers. Test chambers were covered and there was headspace 

above solutions. Test solutions were not aerated and there were three replicates per treatment. No 

deviations from the method guidance. 

 Number of replicates, fish per replicate: Three replicates 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, pH 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 from 6.2 – 6.8 mg/L, pH ranged from 8.3 – 8.4 s.u. 

- Test temperature range: 25 ± 2oC (actual measurements ranged from 23.0 to 25.1oC). No 

deviations or differences from method guidance. 

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

Arithmetic mean. No deviations or differences from method guidance. 

RESULTS

 Nominal concentrations (as mg/L): 0 (Solvent control), 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. LC50, LCo, LL50, or LL0 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 
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 Remarks field for Results. Discuss if element effect concentration is greater than materials 

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

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% / Dilution water control: 0%

- Abnormal responses: None 

- Reference substances (if used)- results: None 

- Any observations, such as precipitation that might cause a difference between measured and 

nominal values: Due to the low solubility of PETN in 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 solution. The low solubility can account for the difference 

between the nominal and measured concentrations. 

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 P. promelas 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-, 48-, 72-, and 96-hour EC50 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 highest 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 (PETN) to 

the Fathead Minnow (Pimephales promelas) 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 

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 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. Fish Acute Toxicity Test, Freshwater and Marine. OPPTS 850.1075. Ecological Effects 

Test Guidelines. EPA 712-C-96-118. 

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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 Remarks field for General Remarks (Use for any other comments necessary for clarification.)