SRC TR-14-010

HPV2 TEST RULE:

RESPONSE TO AECOM COMMENTS DATED MARCH 5, 2014

(FINAL)

1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester), CASRN 78-11-5

Prepared by:

Amy Nguyen, Jennifer Rayner, Teresa Manyin, and William Richards

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

April 22, 2014
HPV2 Test Rule: Response to AECOM (Final) CASRN 78-11-5

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On March 5, 2014, AECOM submitted a memo to EPA1regarding difficulties in maximizing the 

solubility of CASRN 78-11-5 (1,3-propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester);

pentaerythritol tetranitrate [PETN]) for the acute aquatic toxicity studies to fish and Daphnia 

required in the final test rule, “Testing of Certain High Production Volume Chemicals; Second 

Group of Chemicals” (76 FR 1067, January 7, 2011; referred to herein as HPV2 test rule; 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-

0531-0579 as of April 22, 2014). The memo was submitted in response to EPA’s data adequacy 

review for CASRN 78-11-5 (available online at http://www.regulations.gov#!documentDetail;

D=EPA-HQ-OPPT-2009-0112-0211 as of April 22, 2014), which determined that the acute 

toxicity studies of fish and Daphnia that were previously submitted to EPA by the sponsor, Dyno 

Nobel Inc., on March 26, 2013 (submission available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0713 as of April 22, 2014) were not adequate 

to satisfy the C4 aquatic toxicity testing requirement in the HPV2 test rule. The memo addressed 

some study deficiencies, reported the results of tests examining the effects of various factors on 

the solubility of CASRN 78-11-5, and presented possible options for performing the toxicity

tests to fulfill test rule requirements.

The comments and suggestions from AECOM are addressed below:

(1) Comment: AECOM had cited the use of EPA SW-846 Method 8330A (Nitroaromatics and 

Nitramines by High Performance Liquid Chromatography) for analytical determination of PETN

concentrations in the previously submitted acute aquatic toxicity tests (Naddy, 2012, 2013). In 

its March 5, 2014 memo, AECOM clarified that a wavelength of 215 nm was used for the 

detection of PETN, rather than the wavelength of 254 nm specified in Method 8330A. AECOM 

noted that the update of this method (8330B; Nitroaromatics, Nitramines, and Nitrate Esters by 

High Performance Liquid Chromatography) added PETN to the analyte list. In response to 

EPA’s comment that chromatograms and limit of detection (LOD) values were not included in 

the study reports for the acute aquatic toxicity tests, AECOM stated that this information is

provided by the analytical laboratory only in higher level study reports.

EPA’s response: EPA considers the analytical method used in the acute toxicity to fish and 

Daphnia tests (i.e., EPA SW-846 Method 8330A/B with a wavelength of 215 nm2) acceptable 

for measurement of PETN concentrations in these tests and any future aquatic toxicity studies. 

EPA continues to consider the absence of LOD values and chromatograms in the study reports to 

be deficiencies. EPA requests that the sponsor provide sample chromatograms and the LOD

value(s) of the analytical method in all subsequent study reports. 

(2) Comment: AECOM noted that the purity of the test substance, which was not provided in the 

original study reports, was 98.43%.

 

1 Memo addressed to Mr. John Schaeffer of EPA from Dr. Rami B. Naddy of AECOM, the performing laboratory 

for aquatic toxicity studies on fish and Daphnia that were previously submitted by Dyno Nobel Inc.

2 EPA SW-846 Method 8330B recommends a similar wavelength (214 nm) for detection of PETN. EPA SW-846 

Methods 8330A and 8330B are available online at http://www.epa.gov/osw/hazard/testmethods/sw846/pdfs/

8330a.pdf and http://www.epa.gov/osw/hazard/testmethods/pdfs/8330b.pdf, respectively, as of April 22, 2014.
HPV2 Test Rule: Response to AECOM (Final) CASRN 78-11-5

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EPA considers the purity of 98.43% reported in the March 5, 2013 submission to be acceptable 

for the previous acute toxicity to fish and Daphnia tests and any future aquatic toxicity tests. In 

all future study reports submitted to EPA, the sponsor should provide the test substance purity 

and the complete composition of the test material, including the percentages of all impurities.

(3) Comment: AECOM states “Additional analytical samples were not measured in the 1.0 mg/L 

nominal PETN treatment for the fish because this was considered an extra treatment (i.e. the 

nominal 2.0 mg/L treatment was at the solubility limit for PETN and the idea was that if we 

could demonstrate no toxicity at that level, the lower treatments were not as important.”

EPA’s response: It was unclear from the acute toxicity to fish study report (Naddy, 2013) that 

the definitive test was intended to be a limit test (i.e., a test conducted at the limit of solubility to 

demonstrate no toxicity at this concentration); if that was the case, then the use of an 

intermediary concentration of 1.0 mg/L was not necessary. OPPTS 850.1075, p. 8, Section (f) 

“Performance of the test”, Sub-section (2) “Test concentrations”, item (v) “Concentration 

analysis”, part (C) states: “In static-renewal tests, the test substance should be measured in each 

replicate at the beginning and end of test and just before and after each renewal.” For future 

studies, analysis of test substance concentrations should be performed on all replicates included 

in the study and should include samples taken just prior to (“old” test solutions) and after (“new” 

test solutions) each renewal for each replicate. 

(4) Comment: Regarding the fact that the acute toxicity to fish study report (Naddy, 2013) was 

missing experimental details for the range-finding (preliminary) test #2, AECOM states that the 

data sheets for the preliminary studies were included in Appendix D of the report.

EPA’s response: According to the study report, two preliminary tests were conducted, the first 

in November 2011 and the second in November 2012. Appendix D of the study report only 

includes the detailed results from the first (November 2011) preliminary test. The results of the 

second (November 2012) preliminary test are briefly summarized on page 11 of the study report, 

but details are not provided.

(5) Comment: AECOM states that the concentration of PETN in the acetone stock solution (used 

in the definitive test) was “confirmed at 5,400 mg/L”.

EPA’s response: EPA appreciates this additional information, which demonstrates that the 

actual concentration of the stock solution was similar to the nominal concentration of 

5000 mg/L.

(6) Comment: AECOM proposes the use of methanol or acetone in test solutions at

concentrations in excess of the maximum solvent concentration recommended by standard 

aquatic toxicity testing guidelines to achieve target PETN test concentrations for the acute fish 

and Daphnia toxicity studies.

EPA’s response: EPA does not recommend the use of methanol, acetone, or other solvents at 

concentrations exceeding the maximum concentration recommended by aquatic toxicity test 
HPV2 Test Rule: Response to AECOM (Final) CASRN 78-11-5

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guidelines. Specifically, solvent concentrations may not exceed 0.5 mL/L, as stated in ASTM 

E729-96 (test rule-specified guideline for acute toxicity to fish and invertebrates). 

Recommendations for Further Testing to Satisfy Aquatic Toxicity Testing Requirements:

The sponsor should first perform a study to determine the solubility of CASRN 78-11-5 in the 

fish and Daphnia test medium.

1

 The solubility study should be performed under similar 

conditions (e.g., temperature, test medium, concentration of organic solvent, etc.) to those used 

in the aquatic toxicity tests. To conduct this solubility study, EPA recommends that the sponsor 

contract the same laboratory (U.S. Army Center for Health Promotion and Preventative Medicine

[CHPPM]) that conducted the solubility test in deionized water (Hable et al., 2008). The sponsor 

should report the results of this solubility study to EPA. If the measured solubility limit of 

CASRN 78-11-5 in the fish and Daphnia test medium is less than or approximates the highest 

mean measured concentration in the fish and daphnid studies (i.e., 1218 μg/L in the fish test and 

943 μg/L in the daphnid test), then EPA will accept the previously submitted toxicity studies in 

fish and daphnids (Naddy, 2012, 2013) as adequate and no further testing is needed. If the 

measured solubility limit of CASRN 78-11-5 in the fish and Daphnia test medium exceeds the 

highest mean measured concentration in the fish and daphnid studies, the sponsor will need to

repeat the fish and daphnid studies. 

If additional studies in fish and daphnids are needed, EPA provides the following 

recommendations for conducting adequate studies:

(1) Since the previously submitted toxicity to algae study (Pillard, 2008) was able to achieve test 

substance concentrations that approximated the water solubility limit of the chemical, EPA 

recommends that a similar strategy be used for preparation of test solutions for the additional fish 

and daphnid toxicity studies.2 For the algae study, the testing laboratory received an 

approximately saturated solution of PETN dissolved in water (nominal concentration of 

~2000 μg/L; measured concentration of 2850 μg/L) from CHPPM and added macro- and micronutrients (salts) to this solution to prepare the highest test concentration. Mean measured test 

concentrations during the 96-hour algae test were 3.2, 30, 310, and 2850 μg/L for nominal 

concentrations of 2, 20, 200, and 2000 μg/L, respectively, indicating that the laboratory was 

successful in preparing test concentrations that approximated the water solubility limit of PETN 

and that measured concentrations were similar to nominal concentrations. 

To conduct the fish and daphnid toxicity studies, EPA suggests that CHPPM prepare a saturated 

solution of PETN in test medium and ship this solution to AECOM. The saturated solution 

would be used as the highest test concentration and could be diluted with test medium to attain 

lower test concentrations. Alternatively, CHPPM could send AECOM a saturated solution of 

PETN in distilled water, as was done for the algae test. In this case, AECOM would need to add 

 

1 The test media used in the fish and daphnid studies (Naddy, 2012, 2013) appear to have been prepared using 

identical methods. Therefore, only one solubility test needs to be performed to determine solubility in the test 

medium; the results will be applicable to both the fish and daphnid tests.

2 AECOM stated “When we used this same technique [as used in the algae study] for the fish/invertebrate studies, it 

resulted in very low levels of PETN in solution, ~18 μg/L.” However, it is not clear exactly what technique was 

used.
HPV2 Test Rule: Response to AECOM (Final) CASRN 78-11-5

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salts to the stock solution to achieve a hardness level that falls within test guideline 

recommendations. For the algae study, the temperature of the saturated solution was 

incrementally adjusted to 30 oC before salts were added and the solution was diluted; a similar 

technique may be used for the fish and daphnid studies to aid in the dissolution of salts and 

solubilization of the test substance in solution. 

If shipment volume is a concern, the sponsor could consider performing the fish and daphnid 

toxicity tests with no renewal (i.e., static conditions). In the previously submitted static toxicity 

to algae test (Pillard, 2008), concentrations were 82-90% of initial concentrations over the 96-

hour test period, suggesting that the test material was stable for at least 96 hours in algae test 

medium. 

Prior to conducting static fish and daphnid tests, EPA recommends that the sponsor conduct 

preliminary tests to determine whether test substance concentrations can be satisfactorily 

maintained over the test period (96 hours for fish and 48 hours for Daphnia) in the test medium. 

If test concentrations are reasonably maintained over the test period, the required aquatic toxicity 

tests could be performed under static conditions. EPA adds that any future aquatic toxicity tests 

in fish and Daphnia should be conducted with analytical monitoring every 24 hours to 

characterize test concentrations throughout the test period.

Test solutions in the exposure vessels should be thoroughly stirred to assure homogeneity before 

organisms are added to the vessels. The procedures for mixing the concentrations should be 

adequately documented in the study reports. EPA notes that the study reports for the submitted 

acute toxicity to fish (Naddy, 2013) and acute toxicity to Daphnia (Naddy, 2012) tests did not 

adequately describe the procedures for mixing test solutions. Therefore, it is unknown if test 

solutions reached homogeneity prior to analytical sampling. If the test substance was not 

homogeneously distributed in the test vessels at study initiation, this may have contributed to the 

increases observed in measured concentrations during the 24-hour renewal periods. 

(2) If the techniques recommended above are not successful in producing consistent PETN 

concentrations up to the solubility limit in test medium, EPA recommends that the sponsor

attempt to prepare a more concentrated (> 5000 mg/L) stock solution of CASRN 78-11-5 in 

acetone. One of the possible options presented in AECOM’s memo involved the use of higher 

target concentrations (greater than 2000 or 2500 μg/L) in test solutions to achieve higher PETN 

concentrations; since the preparation of higher target test solution concentrations with a stock 

solution of 5000 mg/L in acetone would result in an acetone concentration greater than 0.5 mL/L 

in test solutions, AECOM requested permission to exceed the recommended solvent limit. EPA 

agrees with AECOM that the use of target nominal test solution concentrations greater than (e.g., 

2-fold) the solubility limit of PETN in water may allow AECOM to attain a concentration that 

approximates the water solubility limit of PETN (experimentally determined to be 2130 μg/L at 

20 °C by Hable et al., 2008). As stated in OECD’s Guidance Document on Aquatic Toxicity 

Testing of Difficult Substances and Mixtures (OECD, 2002; p. 20, Section 3 “Media preparation 

and exposure systems for difficult test substances”): “...While the target should be to achieve 

exposure concentrations up to the aqueous solubility of the test substance, it may be necessary to 

utilize higher nominal concentrations to achieve it.” However, EPA does not recommend that 

AECOM exceed an acetone concentration of 0.5 mL/L in test solutions. 
HPV2 Test Rule: Response to AECOM (Final) CASRN 78-11-5

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The nominal concentration of PETN in the stock solution used in the fish and Daphnia acute 

toxicity tests (Naddy, 2013 and Naddy, 2012, respectively) was ~5000 mg/L in acetone; test 

solutions prepared from this stock solution with a nominal concentration of 2500 μg/L contained 

a solvent concentration of 0.5 mL/L. If a stock solution of 7000 or 8000 mg/L in acetone could 

be prepared, then test solutions with nominal concentrations of 3500 or 4000 μg/L, respectively, 

could be made from this stock solution without exceeding a solvent concentration of 0.5 mL/L. 

Prior to implementing this strategy for the fish and daphnid tests, EPA recommends that the 

sponsor conduct tests to determine if (1) a stock solution of 7000 or 8000 mg/L in acetone can be 

prepared successfully (including analytical verification of the concentration of PETN in the stock 

solution) and (2) if this stock solution can be used to prepare test concentrations that approximate 

the solubility limit of PETN in the test medium without exceeding an acetone concentration of 

0.5 mL/L. Recommendations for achieving homogeneity of test solutions (described above) 

would also apply to this approach.

REFERENCES

ASTM. 2007. E729-96: Standard Guide for Conducting Acute Toxicity Tests on Test Materials 

with Fishes, Macroinvertebrates, and Amphibians. 

Hable, M.A., Oliver, C.G., and Johnson, M.S. 2008. Determination of the water solubility, 

octanol-water partition coefficient and biodegradation potential of pentaerythritol-tetranitrate 

(PETN). Performing Laboratory: U.S. Army Center for Health Promotion and Preventative 

Medicine, Aberdeen Proving Ground, MD. Project ID 82-XE-07TY-08. Sponsor: Dyno Nobel, 

Inc., Salt Lake City, UT. Study report available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0062 as of April 22, 2014.

Naddy, R. 2012. Acute toxicity of pentaerythritol tetranitrate (PETN) to the cladoceran 

(Daphnia magna) under static-renewal test conditions. Performing Laboratory: AECOM Fort 

Collins Environmental Toxicology Laboratory, Fort Collins, CO. Project ID 60225262-304-010. 

Sponsor: Dyno Nobel, Inc., West Jordan, UT. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0713 as of April 

22, 2014.

Naddy, R. 2013. Acute toxicity of pentaerythritol tetranitrate (PETN) to the fathead minnow 

(Pimephales promelas) under static-renewal test conditions. Performing Laboratory: AECOM 

Fort Collins Environmental Toxicology Laboratory, Fort Collins, CO. Project ID 60279946-

100-020. Sponsor: Dyno Nobel, Inc., West Jordan, UT. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0713 as of April 

22, 2014.

OECD. 2002. “OECD Series on Testing and Assessment Number 23: Guidance document on 

aquatic toxicity testing of difficult substances and mixtures”. Available online at 

http://www.oecd-ilibrary.org/environment/guidance-document-on-aquatic-toxicity-testing-ofdifficult-substances-and-mixtures_9789264078406-en as of April 22, 2014.
HPV2 Test Rule: Response to AECOM (Final) CASRN 78-11-5

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Pillard, D.A. 2008. Toxicity of pentaerythritol tetranitrate (PETN) to the unicellular green alga 

(Selenastrum capricornutum) under static test conditions. Performing Laboratory: ENSR Fort 

Collins Environmental Toxicology Laboratory, Fort Collins, CO. Project ID 08503-131-073-

002. Sponsor: Dyno Nobel, Inc., Salt Lake City, UT. Study report available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2005-0033-0294 as of April 

22, 2014.

US EPA. 1996. Ecological Effects Test Guidelines: OPPTS 850.1075, Fish Acute Toxicity 

Test, Freshwater and Marine. Available online at http://www.epa.gov/ocspp/pubs/frs/home/

draftguidelines.htm as of April 22, 2014.