SRC TR-13-057

HPV2 TEST RULE DATA ADEQUACY REVIEW (REVISED FINAL):

ACUTE TOXICITY TO FISH AND DAPHNIA

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

Prepared by:

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

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
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission from Dyno Nobel, Inc. (dated March 26, 2013) that 

contains data from toxicity testing of 1,3-propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate

(ester) (CASRN 78-11-5; synonym: pentaerythritol tetranitrate or PETN) and was received by 

EPA in response to 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; 

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

0531-0579 as of April 16, 2014). 

The HPV2 test rule requires the following toxicological testing for CASRN 78-11-5: C4 

(consisting of acute toxicity to fish and acute toxicity to Daphnia testing if log Kow < 4.2 or 

chronic toxicity to Daphnia testing if log Kow ≥ 4.2). Since the log Kow of CASRN 78-11-5 has 

been experimentally determined to be 2.041, which is < 4.2, the C4 aquatic toxicity testing 

requirement is considered to include acute toxicity to fish and acute toxicity to Daphnia testing.

The March 26, 2013 submission from Dyno Nobel, Inc. (available online at 

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

16, 2014) contained the following study reports, which were submitted in response to the C4 

testing requirement: 

 Acute Toxicity of Pentaerythritol Tetranitrate (PETN) to the Fathead Minnow 

(Pimephales promelas) under Static-Renewal Test Conditions 

 Acute Toxicity of Pentaerythritol Tetranitrate (PETN) to the Cladoceran (Daphnia 

magna) under Static-Renewal Test Conditions 

These newly submitted data are evaluated for adequacy herein. No other toxicity data are 

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

The submitted data are evaluated for adequacy in Section D. Each study was summarized 

according to a standard format and assigned a Klimisch reliability code based on its scientific 

merit and conduct, using professional judgment. To determine whether data were adequate to 

satisfy SIDS endpoints, study details were compared to current standard test guidelines to 

establish whether the study met current testing requirements. If the data available for the 

endpoint were determined to be inadequate, a basis for that conclusion is supplied. 

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Data submitted by Dyno Nobel, Inc. on March 26, 2013 are not considered adequate to satisfy 

the C4 testing requirement for CASRN 78-11-5 promulgated in the final HPV2 test rule. Both 

the acute toxicity to fish study and the acute toxicity to Daphnia study are considered unreliable 

because the concentrations tested were less than the solubility limit of the test item and the tests 

 

1 The log Kow of PETN in water was reported in a letter of October 15, 2008 and its attached partition coefficient

study report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0062 as of April 16, 2014).
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

2

failed to identify an LC50 or EC50 for fish and daphnids, respectively. The mean measured test 

substance concentration at the highest concentration tested was 1218 and 943 μg/L, respectively, 

for the fish and Daphnia studies, compared to the reported water solubility limit of 2130 μg/L at 

20°C

1

. Furthermore, a previously submitted study of the toxicity of PETN to algae (Pillard 

2008)2achieved a mean measured test substance concentration of 2850 μg/L, suggesting that the 

highest concentrations tested in the fish and daphnid studies may have been as low as one half to 

one third of the potential solubility limit. In a cover letter attached to the acute toxicity to fish 

study, the study director speculates that the maximum measured PETN concentration in the fish 

and daphnid tests was less than the reported water solubility limit of 2130 μg/L due to “increased 

polarity of the water used in the toxicity studies (i.e., the required laboratory test water has a 

target water hardness and alkalinity of 170 and 115 mg/L as CaCO3, respectively).” However, 

the study reports do not provide experimental data examining the relationship between solubility 

of PETN and water hardness and/or alkalinity to support this statement.

Additional deficiencies of the acute toxicity to fish and acute toxicity to Daphnia tests include 

the following:

(1) The acute toxicity to fish and daphnid test reports did not explain why measured 

concentrations of PETN showed an apparent increase from the beginning to the end of 

the 24-hour renewal intervals. 

(2) OPPTS 850.1075 (test guideline cited in the acute toxicity to fish study report) requires 

analysis of test item concentrations for replicate samples at the start and end of the test, as 

well as just before (“old” test solutions) and after (“new” test solutions) each renewal in 

static-renewal tests. However, measurements in the acute toxicity to fish test were not 

performed on “old” samples collected on days 2 or 4 for the nominal concentration of 

1000 μg/L. In addition, the “old” test solution from only a single replicate (of 3 

replicates) was analyzed on days 1 and 3 for the nominal concentration of 1000 μg/L.

(3) OPPTS 850.1010 (test guideline cited in the acute toxicity to Daphnia study report) 

requires that measured concentrations not vary by more than 20% among replicate test 

chambers. However, variation in test substance concentration among the replicates in the 

acute toxicity to daphnid test for “old” samples (taken just before each renewal with fresh 

medium) collected on day 2 was greater than 20%. 

(4) Details regarding the analytical measurements of test substance concentrations were

missing from the fish and daphnid study reports, including the limit of detection (LOD)

for the analytical method and chromatograms for the analytical measurements. Also, the 

fish study report was missing experimental details for range-finding test 2. 

Conclusion: The fish and daphnid studies should be repeated to assure that PETN is tested up to 

its solubility limit in a test medium appropriate for fish and Daphnia. The water solubility study 

report for this compound (available online at http://www.regulations.gov/#!documentDetail;

 

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study 

report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0062 as of April 16, 2014).

2 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 16, 2014.
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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D=EPA-HQ-OPPT-2007-0531-0062 as of April 16, 2014) stated that “the process for PETN 

equilibration in aqueous solutions at room temperature is slow” based on the achievement of 

maximum solubility after 48 hours or longer; therefore, the laboratory should ensure that 

sufficient time is allowed for mixing of test solutions. It is recommended that the study design 

for further acute toxicity testing in fish and Daphnia includes the conduct of preliminary testing 

to determine what procedures are necessary to assure that PETN reaches maximum possible 

solubility in the intended test medium (i.e., dilution water) at each concentration that is to be 

tested in the main toxicity study. In addition, the study report should include the chromatograms 

and LOD for the analytical measurements to allow an independent assessment of the validity of 

the method used for quantitation of this test substance.
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Fish

Summary of endpoint: Not Adequate

Study 1: 96-hour acute toxicity study in fathead minnow (Naddy, 2013) 

Title: Acute toxicity of pentaerythritol tetranitrate (PETN) to the fathead minnow (Pimephales 

promelas) under static-renewal test conditions

This study is: Adequate Not Adequate

This study is considered unreliable because the test was not conducted up to the solubility limit 

of the test item and the test failed to identify an LC50 for fish. The mean measured concentration 

of test item of 1218 μg/L at the highest concentration tested is approximately 2-fold lower than 

the reported water solubility of 2130 μg/L at 20°C

1

.

TEST SUBSTANCE 

Identity: 1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester); referred to as 

pentaerythritol tetranitrate (PETN) in study report; CASRN 78-11-5 (synonyms: penthrit; 

pentrite; nitropenta; nitropentaerytritol) 

Remarks: Received as a stock solution in acetone with a nominal concentration of 5000 mg 

PETN/L and a measured concentration of 5400 mg PETN/L2; clear, colorless liquid; purity:

98.43%3. 

METHOD

Method/guideline followed: OPPTS 850.1075 (“Fish Acute Toxicity Test, Freshwater and 

Marine”) and ASTM E729-96 (“Standard Guide for Conducting Acute Toxicity Tests on Test 

Materials with Fishes, Macroinvertebrates, and Amphibians”)

Test type: Acute fish toxicity test (static-renewal)

GLP compliant?: Yes, with the exception that “analytical data were generated following 

NELAP (National Environmental Laboratory Accreditation Conference) guidelines.” [Naddy 

2013, p. iv]

 

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study 

report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0062 as of April 16, 2014).

2 The measured concentration of the stock solution was reported in a memo, dated March 5, 2014, from Dr. Rami B. 

Naddy of AECOM to Mr. John Schaeffer of EPA.

3 The purity of the test substance was reported in a memo, dated March 5, 2014, from Dr. Rami B. Naddy of 

AECOM to Mr. John Schaeffer of EPA.
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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Year study was performed: 2012

Test species: Fathead minnow (Pimephales promelas) obtained from in-house laboratory stocks

Analytical monitoring: Yes (definitive test only)

Exposure duration: 96 hours 

Concentrations tested: 

Preliminary test 1 (nominal): 0, 100, 500, and 2000 μg/L

Preliminary test 2 (nominal): 0, 1000, and 2000 μg/L

Definitive test: Nominal concentrations: 0, 1000, and 2000 μg/L; mean measured concentrations:

0, 723, and 1218 μg/L. Mean measured concentrations were based on the average of newly 

prepared test solutions (“new” samples) and test solutions collected at the time of renewal (“old” 

samples). New samples were collected for analysis on days 0, 1, 2, and 3 and old samples were 

collected for analysis on days 1, 2, 3, and 4. 

Test method/conditions remarks:

Test Species: 

Fathead minnow (Pimephales promelas); 14-days old at initiation; obtained from in-house 

laboratory stocks; mean length of 8.5 mm and mean weight of 3.4 mg at initiation. Mean length 

and weight were based on 10 fish from the same batch as those used for the test. Fish were 

acclimatized to test conditions for at least 7 days prior to exposure and were not fed in the 48 

hours prior to initiation of exposure or during the study. 

Test Item and Test Item Preparation:

The test item was received as a stock solution in acetone with a nominal concentration of 

5000 mg PETN/L (measured concentration of 5400 mg PETN/L1) from the United States Army 

Center for Health Promotion and Preventative Medicine (CHPPM). The stock solution was 

received and stored in an amber jar at 4°C and was rewarmed to room temperature prior to use. 

“The test substance was assumed to be 100% active ingredient for purposes of stock solution and 

exposure concentrations calculation and preparation.” [Naddy 2013, p. 2]

“Each day, the highest test concentration, 2000 μg PETN/L (nominal), was prepared by addition 

of 0.8 mL of the PETN stock solution in acetone... [nominal concentration of 5000 mg PETN/L]

to 2 L of dilution water. A 650 mL aliquot of the highest concentration was then diluted to 1.3 L 

to prepare the 1000 μg PETN/L (nominal) treatment daily. This was sufficient volume for 

biological and analytical sampling.” [Naddy 2013, p. 4]

The dilution water, which was periodically analyzed for contaminants, was 100% Milli-Q

® water 

(ASTM type I; Millipore, Billerica MA) that was adjusted to a target hardness of 170 ± 10 mg/L 

as CaCO3 and alkalinity of 115 ± 5 mg/L as CaCO3. At test initiation, the dilution water had a 

pH of 8.3, with hardness and alkalinity (as CaCO3) of 174 and 115 mg/L, respectively. Total 

 

1 The measured concentration of the stock solution was reported in a memo, dated March 5, 2014, from Dr. Rami B. 

Naddy of AECOM to Mr. John Schaeffer of EPA.
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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residual chlorine was < 0.02 mg/L and total ammonia (as N) was < 1.0 mg/L. Conductivity was 

593 μS/cm and resistance was 18 MΩ·cm.

Test Procedures:

Preliminary Test Procedure: Two preliminary, 96-hour tests were conducted using a stock 

solution of 1000 mg PETN/L in methanol. Nominal concentrations in test 1 were 0, 100, 500, 

and 2000 μg/L. Nominal concentrations in test 2 were 0, 1000, and 2000 μg/L. Analytical 

monitoring was not conducted in the preliminary studies, although analytical monitoring was 

conducted on test substance solutions that were similarly prepared. For preliminary test 1, two 

replicates, with 5 fish per replicate, were tested. Test conditions were similar to those in the 

definitive test, except that test organisms were 4 days old at the start of exposure and media were

renewed only once (at 48 hours) during the 96-hour exposure. No experimental details were 

reported for preliminary test 2.

Definitive Test Procedure: Juvenile fish (10/replicate; 3 replicates/concentration) were exposed 

to the test item under static-renewal conditions for 96 hours; test solutions were renewed once 

daily at the beginning of each 24-hour period. Fish loading rate was ≤ 0.5 g/L of test medium.

Solvent (0.4 mL acetone/L dilution water) and dilution water controls were exposed to the same 

conditions in the absence of the test item. 

“Test chambers were new, 500-mL (nominal volume) plastic cups containing ~250 mL of test

solution... Test chambers were covered during the test to minimize evaporation and possible airborne contamination.” [Naddy 2013, p. 4]

“The exposure chambers were housed in a temperature-controlled environmental chamber that

maintained the test solution temperature at 25 ± 2 °C. Lighting was controlled to provide 16

hours of light per 24-hour period; light intensity was ambient (approximately 50 - 100 

footcandles).” [Naddy 2013, p. 5]

Fish were observed daily for survival. A fish was considered dead if it was non-responsive to 

gentle prodding. Following euthanasia, length measurements were recorded for all surviving fish 

in the control and treatment groups. Fish weight was recorded for control groups only.

Hardness and alkalinity (both as mg CaCO3/L), total ammonia (as mg N/L), and total residual 

chlorine were recorded at initiation in the controls and the highest test item concentration. 

Dissolved oxygen, pH, and conductivity were recorded for one replicate at initiation and daily 

thereafter. Temperature was recorded for each replicate and concentration throughout the study.

For analytical monitoring, “samples for PETN analysis were collected from bulk solutions at test 

initiation, prior to distributing the test solutions to the test chambers, from each freshly prepared 

test treatment, the solvent control, and the dilution water. On days 1, 2, and 3, samples were 

collected from bulk solutions of all freshly prepared test treatments after distributing to test 

chambers, as well as from all replicates of old test solutions for treatments after organisms had 

been transferred to new solutions. Only one replicate of old test solutions was analyzed on days 

1 and 3... [for the nominal concentration of 1000 μg/L. None of the day 2 or day 4 old test 

solution samples were analyzed for the 1000 μg/L concentration]... Each replicate of the old test 
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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solutions was sampled from all treatments at test termination after removing the test organisms...

All samples were poured directly into certified clean 500-mL glass amber bottles. The samples 

were refrigerated until shipped (on ice) [to an independent laboratory for analysis].” [Naddy 

2013, p. 6]

“EPA Method 8330A was used following USEPA (1986) guidelines to analyze for 

nitroaromatics and nitramines by HPLC [using a wavelength of 215 nm1,2].” [Naddy 2013, p. 6]

The study report did not include the limit of detection (LOD) for the analytical method.

Regarding statistical analyses, “given that there was not ≥ 50% mortality observed in any 

treatment of this study, statistical analyses were not required. The concentrations estimated to 

cause death (or immobility) of 50% of the test organisms after 24, 48, 72, and 96 hours of 

exposure (the 24-, 48-, 72-, and 96-hour LC50, respectively) were determined by inspection. The 

LC50 values are expressed as average measured PETN concentrations measured in new and old 

test solutions (μg PETN/L).” [Naddy 2013, p. 6]

RESULTS

Endpoint value(s): The following endpoint values are based on mean measured concentrations:

96-hour LC50 > 1218 μg/L 

96-hour LOEC (mortality) = 1218 μg/L

96-hour NOEC (mortality) = 723 μg/L

Results remarks:

Preliminary Test Results:

Mortality was observed at the highest (nominal) concentration of 2000 μg/L in both preliminary 

tests. In test 1, mortality percentages at 96 hours were 0%, 0%, 0%, and 60% for the nominal 

concentrations of 0 (dilution water control), 100, 500, and 2000 μg/L, respectively. In test 2, 

mortality percentages at 96 hours were 0%, 0%, 0%, and 70% for the nominal concentrations of 

0 (dilution water control), 0 (solvent control), 1000, and 2000 μg/L, respectively.

The concentrations of test item in media were not measured in the preliminary tests. Analysis of 

solutions prepared similarly to those used in test 1 showed measured concentrations of 

approximately 85% and 86% of nominal values for the nominal concentrations of 500 and 1000 

μg/L, respectively. A sample corresponding to the 2000 μg/L nominal concentration was not 

analyzed.

Definitive Test Results:

 

1 The wavelength of 215 nm was reported in a memo, dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to 

Mr. John Schaeffer of EPA.

2 EPA SW-846 Method 8330B (Nitroaromatics, Nitramines, and Nitrate Esters by High Performance Liquid 

Chromatography), which is the updated version of EPA SW-846 Method 8330A, 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 16, 2014.
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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Mortality data are summarized in Table 1. After 96-hours of exposure, 26.7% mortality was 

observed in the 2000 μg/L group, compared to 0% in the dilution water control and 3.3% in the 

solvent control and 1000 μg/L groups (statistical significance was not reported). No deaths 

occurred in the 1000 or 2000 μg/L groups during the first 72 hours of the exposure period.

Table 1. Mortality (% of total number of fish exposed per group) in fish exposed to PETN 

Nominal 

concentration 

(μg/L)

Observation Time (hr)

0 24 48 72 96

0 (solvent) 0% 0% 3% (1/30) a 3% (1/30) 3.3% (1/30)

0 (dilution water) 0% 0% 0% 0% 0% (0/30)

1000 0% 0% 0% 0% 3.3% (1/30)

2000 0% 0% 0% 0% 26.7% (8/30)

a The number in parentheses is the cumulative number of dead fish per total number exposed.

At the 96-hour observation, concentration-related “weakness” was observed in surviving fish as 

follows: solvent control, 1/29 (3.4%); dilution water control, 0/30 (0%); 1000 μg/L, 7/29 

(24.1%); and 2000 μg/L, 18/22 (81.8%). No weakness was observed from 0 to 72 hours in 

control or test item treatment groups.

Exposure to the test material did not appear to affect fish growth as assessed by length, although 

no summary statistics were reported. 

Test chamber temperatures, measured daily, ranged from 23.0 to 25.1 oC. The pH range was 8.2-

8.4 and the dissolved oxygen range was 6.2-6.9 mg O2/L. Conductivity was 572-631 μS/cm. 

These values “were in compliance with the protocol and ASTM requirements.” [Naddy 2013, p. 

7]

Mean measured concentrations (based on combined “old” and “new” samples) for the 0 (solvent 

control), 0 (dilution control), 1000 μg/L, and 2000 μg/L groups were < LOD, < LOD, 723 μg/L, 

and 1218 μg/L, respectively. As shown in Table 2, measured concentrations (combined new and 

old) for the 1000 and 2000 μg/L groups ranged from 50 to 100% and 49 to 78% of nominal 

concentrations, respectively.
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

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Table 2. Measured Concentrations of PETN in Test Watera

Nominal 

Concentration 

(μg/L)

New Samples Old Samples Mean

Measured

Concentration

(μg/L)b

Study Day

0 1 2 3 1 2 3 4

0 (solvent) < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD

0 (dilution 

water)

< LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD < LOD

1000

530

(53%)

500

(50%)

790

(79%)

820

(82%)

570

(57%)

NAc 1000

(100%)

NA 723

(72%)

2000

1100

(55%)

1000

(50%)

1000

(50%)

1400

(70%)

1167

(58%)

973

(49%)

1533

(77%)

1567

(78%)

1218

(61%)

a Expressed as μg/L; values in () are % of nominal concentration.

b Mean of combined new and old samples.

c NA = Not available; old samples were not measured on study days 2 or 4 for the 1000 μg/L nominal 

concentration.

CONCLUSIONS

The 96-hour LC50 and NOEC (for mortality) for Pimephales promelas were > 1218 μg/L and 

723 μg/L, respectively, based on mean measured concentrations. The LOEC was 1218 μg/L

based on increased mortality, relative to controls, at the highest concentration tested.

STUDY RELIABILITY

[3] Not reliable. This study is considered unreliable because the test was not conducted up to the 

solubility limit of the test item and the test failed to identify an LC50 for fish. The study author 

did not report any precipitation in the test media, indicating that the solubility limit was not 

exceeded. The mean measured concentration of test item of 1218 μg/L at the highest 

concentration tested is approximately 2-fold lower than the reported water solubility of 2130 

μg/L at 20°C

1

. Furthermore, a previously submitted study of the toxicity of PETN to aquatic 

plants (Pillard 2008) achieved a mean measured test substance concentration of 2850 μg/L. 

Given that increased fish mortality was observed at a mean measured concentration of 1218 

μg/L, testing at concentrations up to the solubility limit of 2130 μg/L (or 2850 μg/L based on the 

solubility limit reported in the algae study) may provide sufficient data to determine an LC50

value for PETN in freshwater fish.

Additional deficiencies include the following:

(1) The test report did not explain why measured concentrations of PETN showed an 

apparent increase from the beginning to the end of the 24-hour renewal intervals. 

(2) OPPTS 850.1075 (test guideline cited in the study report) requires analysis of test item 

concentrations for replicate samples at the start and end of the test, as well as just before 

(“old” test solutions) and after (“new” test solutions) each renewal in static-renewal tests. 

However, measurements were not performed on “old” samples collected on days 2 or 4 

 

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study 

report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0062 as of April 16, 2014).
Final HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

10

for the nominal concentration of 1000 μg/L. In addition, the “old” test solution from only 

a single replicate (of 3 replicates) was analyzed on days 1 and 3 for the nominal 

concentration of 1000 μg/L. 

(3) Several experimental details are missing from the study report, including the LOD for the 

analytical method, chromatograms for the analytical measurements, and experimental 

details for range-finding test 2.

REFERENCES

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

16, 2014.

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

16, 2014.

Text in quotes is taken directly from Naddy (2013).
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D-2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Not Adequate

Study 1: Acute toxicity study in Daphnia magna (Naddy, 2012)

Title: Acute toxicity of pentaerythritol tetranitrate (PETN) to the cladoceran (Daphnia magna)

under static-renewal test conditions

This study is: Adequate Not Adequate

This study is considered unreliable because the test was not conducted up to the solubility limit 

of the test item and the test failed to identify an EC50 for daphnids. The mean measured 

concentration of test item of 943 μg/L at the highest concentration tested is approximately 2.2-

fold lower than the reported water solubility of 2130 μg/L at 20°C

1

.

TEST SUBSTANCE 

Identity: 1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester); referred to as 

pentaerythritol tetranitrate (PETN) in study report; CASRN 78-11-5 (synonyms: penthrit; 

pentrite; nitropenta; nitropentaerytritol) 

Remarks: Received as a stock solution in acetone with a nominal concentration of 5000 mg 

PETN/L and a measured concentration of 5400 mg PETN/L2; clear, colorless liquid; purity:

98.43%3.

METHOD

Method/guideline followed: OPPTS 850.1010 (“Aquatic Invertebrate Acute Toxicity Test, 

Freshwater Daphnids”) and ASTM E729-96 (“Standard Guide for Conducting Acute Toxicity 

Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians”)

Test type: Acute toxicity test in daphnids (static-renewal)

GLP compliant?: Yes, with the exception that “analytical data were generated following 

NELAP (National Environmental Laboratory Accreditation Conference) guidelines.” [Naddy 

2012, p. iv]

Year study was performed: 2012

 

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study 

report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0062 as of April 16, 2014).

2 The measured concentration of the stock solution was reported in a memo, dated March 5, 2014, from Dr. Rami B. 

Naddy of AECOM to Mr. John Schaeffer of EPA.

3 The purity of the test substance was reported in a memo, dated March 5, 2014, from Dr. Rami B. Naddy of 

AECOM to Mr. John Schaeffer of EPA.
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Test species: Water flea (Daphnia magna); < 24 hours old; obtained from in-house stocks; 

Batch Number 052912

Analytical monitoring: Yes (definitive test only)

Exposure duration: 48 hours

Concentrations tested: 

Preliminary test (nominal): 0, 268, 419, 547, 809, and 943 μg/L

Definitive test: Nominal concentrations: 0 (solvent control), 0 (dilution water control), 500, 800, 

1100, 1700, and 2500 μg/L; mean measured concentrations: 0 (solvent control), 0 (dilution water 

control), 268, 419, 547, 809, and 943 μg/L. Mean measured concentrations were based on the 

average of newly prepared test solutions (“new” samples) and test solutions collected at the time 

of renewal (“old” samples). New samples were collected for analysis on days 0, 1, and 2 and old 

samples were collected for analysis on days 1, 2, and 3. 

Test method/conditions remarks: 

Test Species: 

Daphnia magna neonates (< 24 hours old) were obtained from AECOM FCETL in-house 

cultures (Batch number 052912). “Organisms were cultured in an environmental chamber at 

25°C, and were moved to a 20oC environmental chamber at least 48 hours prior to test initiation. 

Brood D. magna were also maintained in 100% dilution water for at least 48 hours prior to test 

initiation... Organisms were not fed during testing.” [Naddy 2012, p. 3]

Test Item and Test Item Preparation:

The test item was received as a stock solution in acetone with a nominal concentration of 

5000 mg PETN/L (measured concentration of 5400 mg PETN/L1) from the United States Army 

Center for Health Promotion and Preventative Medicine (CHPPM). The stock solution was 

received and stored in an amber jar at 4°C and was rewarmed to room temperature prior to use. 

“The test substance was assumed to be 100% active ingredient for purposes of stock solution and 

exposure concentrations calculation and preparation.” [Naddy 2012, p. 2]

“The highest test concentration, 2.5 mg PETN/L (nominal), was prepared by addition of the

appropriate volume of the PETN stock solution in acetone to 10 L of dilution water. This was 

sufficient volume for the entire duration of the study. An appropriate volume of the highest 

concentration was then diluted to 1.4 L to prepare all other test solutions daily. This was

sufficient volume for biological and analytical sampling.” [Naddy 2012, pp. 3-4]

The dilution water, which was periodically analyzed for contaminants, was 100% Milli-Q

® water 

(ASTM type I; Millipore, Billerica MA) that was adjusted to a target hardness of 170 ± 10 mg/L 

as CaCO3 and alkalinity of 115 ± 5 mg/L as CaCO3. At test initiation, the dilution water had a 

pH of 8.5, with hardness and alkalinity (as CaCO3) of 172 and 116 mg/L, respectively. Total 

residual chlorine was < 0.02 mg/L and total ammonia (as N) was < 1.0 mg/L. Conductivity was 

579 μS/cm and resistance was 18 MΩ·cm.

 

1 The measured concentration of the stock solution was reported in a memo, dated March 5, 2014, from Dr. Rami B. 

Naddy of AECOM to Mr. John Schaeffer of EPA.
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Test Procedures:

Preliminary Test Procedure: A 48-hour test was conducted using a stock solution concentration 

of 1000 mg PETN/L in methanol. Nominal concentrations were 0 (dilution water control), 100, 

500, and 2000 μg/L. Analytical monitoring was not conducted in the preliminary study, although 

analytical monitoring was conducted on test substance solutions that were similarly prepared. 

Two replicates per group, with 5 organisms per replicate, were tested under similar conditions as 

the definitive test, except that media was not renewed during exposure and a solvent control 

group was not included.

Definitive Test Procedure: Daphnids (5/replicate; 4 replicates per concentration) were exposed to 

the test item at nominal concentrations of 0 (solvent control), 0 (dilution water control), 500, 

800, 1100, 1700, and 2500 μg/L under static-renewal conditions for 48 hours. Test solutions 

were renewed once daily. Daphnid loading was equivalent to 25 daphnids per liter of test 

medium. Solvent (0.5 mL acetone/L dilution water) and dilution water controls were exposed to 

the same conditions in the absence of the test item. 

“Test chambers were 500-mL (nominal volume) plastic cups containing ~200 mL of test 

solution... Test chambers were covered during the test to minimize evaporation and possible airborne contamination.” [Naddy 2012, p. 4] “The exposure chambers were housed in a 

temperature-controlled environmental chamber that maintained the water temperature at 20 ± 

2°C. Lighting was controlled to provide 16 hours of light per 24-hour period; light intensity was 

ambient (approximately 50 - 100 foot-candles).” [Naddy 2012, p. 5]

Daphnids were observed daily for survival. A daphnid was considered dead (immobilized) if it 

was non-responsive to gentle prodding. 

Hardness and alkalinity (both as mg CaCO3/L), total ammonia (as mg N/L), and total residual 

chlorine were measured at initiation in the controls and the highest test item concentration. 

Dissolved oxygen, pH, and conductivity were recorded for one replicate at initiation and daily 

thereafter. Temperature was recorded for each replicate and concentration throughout the study. 

For analytical monitoring, “samples for PETN analysis were collected from bulk solutions at test 

initiation, after distributing the test solutions to the test chambers..., from each freshly prepared 

test treatment, the solvent control, and the dilution water. On day 1, samples were collected from 

bulk solutions of all freshly prepared 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. The samples were refrigerated until 

shipped (on ice) [to an independent laboratory for analysis].” [Naddy 2012, pp. 5-6]
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“EPA Method 8330A was used following USEPA (1986) guidelines to analyze for 

nitroaromatics and nitramines by HPLC [using a wavelength of 215 nm1,2].” [Naddy 2012, p. 6]

The study report did not include the limit of detection (LOD) for the analytical method.

Regarding statistical methods, “given that there was no mortality observed in any treatment of 

this study, statistical analyses were not required. The concentrations estimated to cause death (or 

immobility) of 50% of the test organisms after 24 and 48 hours of exposure (the 24- and 48-hour 

EC50, respectively) were determined by inspection. The EC50 values are expressed as average 

measured PETN concentrations measured in new and old test solutions (μg PETN/L).” [Naddy 

2012, p. 6]

RESULTS

Endpoint value(s): The following endpoint values are based on mean measured concentrations:

48-hour EC50 > 943 μg/L

48-hour LOEC (immobility) > 943 μg/L

48-hour NOEC (immobility) = 943 μg/L

Results remarks:

Preliminary Test Results:

No mortalities or signs of immobility were observed in the preliminary test. The concentrations 

of test item in media were not measured. Analyses of solutions prepared similarly to those used 

in the test showed measured concentrations of approximately 86% of nominal values for the

nominal concentrations of 500 and 1000 μg/L. 

Definitive Test Results:

No mortalities or signs of toxicity were observed in control or test item groups. Test chamber 

temperatures, measured daily, ranged from 19 to 22 oC. The pH was 8.5-8.6. Dissolved oxygen 

was 6.9-7.3 mg O2/L. Conductivity was 563-585 μS/cm. These values “were in compliance 

with the protocol and ASTM requirements.” [Naddy 2012, p. 7]

Measured concentrations are summarized in Table 3. Based on combined “old” and “new”

samples for the 0 (solvent control), 0 (dilution control), 500, 800, 1100, 1700, and 2500 μg/L 

groups, mean measured concentrations were < LOD, < LOD, 268 (54% of nominal), 419 (52% 

of nominal), 547 (50% of nominal), 809 (48% of nominal), and 943 (38% of nominal) μg/L, 

respectively.

 

1 The wavelength of 215 nm was reported in a memo, dated March 5, 2014, from Dr. Rami B. Naddy of AECOM to 

Mr. John Schaeffer of EPA.

2 EPA SW-846 Method 8330B (Nitroaromatics, Nitramines, and Nitrate Esters by High Performance Liquid 

Chromatography), which is the updated version of EPA SW-846 Method 8330A, 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 16, 2014.
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Table 3. Measured Concentrations of PETN Test Watera

Nominal 

concentration 

(μg/L)

New Samples Old Samples Mean

measured 

concentration 

(μg/L)b

Study Day

0 1 1 2

0 (solvent) < LOD < LOD < LOD < LOD < LOD

0 (dilution water) < LOD < LOD < LOD < LOD < LOD

500

240

(48%)

240

(48%)

288

(58%)

305

(61%)

268

(54%)

800

360

(45%)

380

(48%)

463

(58%)

475

(59%)

419

(52%)

1100

480

(44%)

500

(45%)

638

(58%)

570

(52%)

547

(50%)

1700

670

(39%)

620

(36%)

978

(58%)

968

(57%)

809

(48%)

2500

390

(16%)

895

(36%)

1350

(54%)

1138

(46%)

943

(38%)

a Expressed as μg/L; values in () are % of nominal concentration as reported in the study report. 

b Mean of combined new and old samples.

CONCLUSIONS

The 48-hour EC50 and NOEC for Daphnia magna were > 943 μg/L and 943 μg/L, respectively, 

based on mean measured concentrations. The LOEC was > 943 μg/L, based on no effects at the 

highest concentration tested.

STUDY RELIABILITY

[3] Not reliable. This study is considered unreliable because the test was not conducted up to the 

solubility limit of the test item and the test failed to identify an EC50 for daphnids. The study 

author did not report any precipitation in the test media, indicating that the solubility limit was 

not exceeded. The mean measured concentration of test item of 943 μg/L at the highest 

concentration tested is approximately 2.2-fold lower than the reported water solubility of 2130 

μg/L at 20°C

1

. Furthermore, a previously submitted study of the toxicity of PETN to aquatic 

plants (Pillard 2008) achieved a mean measured test substance concentration of 2850 μg/L. 

Additional deficiencies included the following: 

(1) The test report did not explain why measured concentrations of PETN showed an 

apparent increase from the beginning to the end of the 24-hour renewal intervals. 

(2) OPPTS 850.1010 (test guideline cited in the study report) requires that measured 

concentrations not vary by more than 20% among replicate test chambers. However, 

variation in test substance concentration among the replicates for “old” samples (taken 

just before each renewal with fresh medium) collected on day 2 was greater than 20%.

 

1 The solubility of PETN in water was reported in a letter of October 15, 2008 and its attached water solubility study 

report (without appendices) from Mr. Neal Olsen of Dyno Nobel, Inc. to the EPA (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0062 as of April 16, 2014).
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(3) Details regarding the analytical measurements of test substance concentrations were

missing from the study report, including the LOD for the analytical method and

chromatograms for the analytical measurements, .

REFERENCES

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

16, 2014.

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

16, 2014.

Text in quotes is taken directly from Naddy (2012).
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Addendum: Test Guideline Source Material to Support Reliability Ratings Assigned to 

Submitted Studies That Are Considered Inadequate to Satisfy Test Rule Requirements

This addendum provides additional information regarding deficiencies of each of the studies that 

is considered inadequate to satisfy a test rule requirement. For each deviation from relevant test 

guidelines (i.e., standard test guidelines purportedly followed by each study according to the 

study report), quotes of relevant passages from the guidelines are supplied, followed by a brief 

scientific interpretation of the quoted material. Emphasis has been added to quotes (in the form 

of italics) by the reviewer.

In the ASTM E729-96 guideline, p. 3, Section 3 “Terminology”, Sub-section 3.6, the words 

“must” and “should” have very specific meanings: “‘Must’ is used to express an absolute 

requirement, that is, to state that the test ought to be designed to satisfy the specified condition, 

unless the purpose of the test requires a different design. ‘Must’ is only used in connection with 

factors that directly relate to the acceptability of the test... ‘Should’ is used to state that the 

specified condition is recommended and ought to be met if possible. Although violation of one 

‘should’ is rarely a serious matter, violation of several will often render the results questionable.”

Study 1: Acute Toxicity to Fish (Naddy 2013)

Relevant Guidelines: Study was conducted according to OPPTS 850.1075 and ASTM E729-96 

[A] Deviation 1: This study failed to identify an LC50 because mortality at the highest test 

concentration was less than 50% in the definitive test. The study only determined that the LC50

was greater than 1218 μg/L (i.e., 1.218 mg/L), which is considerably less than the reported water 

solubility limit of the test substance (2130 μg/L at 20°C).

OPPTS 850.1075, p. 1, Section (c) “Principle of the test”, Sub-section (1) “Definitive 

test” states: “The goal of the definitive test is to determine concentration-response curves for 

fish mortality, the LC50’s, and the 95 percent confidence intervals for each species tested at 

24, 48, 72, and 96 h in a static, static-renewal, or flow-through system.”; and Sub-section (2) 

“Range-finding or limit testing” states: “Definitive testing may be waived if limit testing 

with at least 30 organisms shows LC50 levels to be greater than 1,000 mg/L based on 100 

percent active ingredients (AI), or the limits of water solubility or dispersibility.”

OPPTS 850.1075, p. 10, Section (g) “Data and reporting”, Sub-section (2)(i) “Test 

report” states: “The test report must include the following:”; and item (viii) states: “The 

LC50 concentration-response curves, LC50 values, and associated 95 percent CI should be 

determined for 24, 48, 72, and 96 h, whenever sufficient data exist.”; and item (xi) states: “If 

no LC50 value is determined, but it can be demonstrated that the concentrations tested were 

the highest possible due to the test chemical’s aqueous solubility limit, self-dispersibility 

limit, or other physicochemical limitations, then the data will be considered for acceptance.”

ASTM E729-96, p. 9, Section 9 “Test Material”, Sub-section 9.3 “Test 

Concentration(s)”, item 9.3.2 states: “In some (usually regulatory) situations, it is necessary

only to determine (1) whether a specific concentration of test material is acutely toxic to the 

test species, or (2) whether the LC50, EC50, or IC50 is above or below a specific concentration. 

For example, the specific concentration might be the concentration occurring in a surface 

water, the concentration resulting from the direct application of the material to a body of 

water, or the solubility limit of the material in water. When there is interest only in a specific 
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concentration, it is often necessary only to test that concentration (see 11.1.1.2) and it is not 

necessary to actually determine the LC50, EC50, or IC50.”

Interpretation: A primary objective of an acute toxicity to fish test is to determine a discrete

LC50 value. If a discrete LC50 value is not determined because insufficient (< 50%) mortality 

occurs at the highest concentration tested and this concentration is below the aqueous solubility 

or dispersibility limit, the test is not considered acceptable. 

[B] Deviation 2: Measured concentrations increased from the beginning to the end of the 24-

hour renewal intervals, but the study report did not provide a valid explanation.

OPPTS 850.1075, p. 2, Section (c) “Principle of the test”, Sub-section (3) “Information 

on the test substance” states: “... A reliable analytical method for quantification of test 

substance concentrations must be available.”

OPPTS 850.1075, p. 2, Section (d) “Validity of the test”, Sub-section (4) states: “... 

[T]here must be evidence that test concentrations remained at least 80 percent of the nominal 

concentrations throughout the test or that mean measured concentrations are an accurate 

representation of exposure levels maintained throughout the test period.

OPPTS 850.1075, p. 10, Section (g) “Data and reporting”, Sub-section (2)(i) “Test 

report” states: “The test report must include the following:” and item (xiii) states: “Any 

protocol deviations or occurrences which may have influenced the final results of the test.”

ASTM E729-96, p. 18, Section 15 “Report”, Sub-section 15.1 states: “The record of the 

results of an acceptable acute toxicity test should include the following information either 

directly or by reference to available documents:” and item 15.1.3 states: “Anything unusual 

about the test, any deviation from these procedures, and any other relevant information.”

ASTM E729-96, p. 16, Section 11 “Procedure”, Sub-section 11.9.3 “Test Material”, item 

11.9.3.1 states: “... If the test material is not uniformly dispersed in the test chamber in static 

and renewal tests, the whole volume of solution in the test chamber should be (1) used as the 

sample or (2) treated appropriately (for example, by adding acid, base, or surfactant and 

mixing thoroughly) to uniformly distribute the test material before a sample is taken.”

Interpretation: Test concentrations must be determined by a reliable analytical method that 

provides an accurate representation of exposure levels. The report must address any deviations 

from the protocol or occurrences that could impact the results. If the test substance is not 

homogeneously distributed in the fluid in the test chambers, additional measures should be taken 

to ensure that samples collected for analysis are representative of actual mean concentrations of 

test substance in the test chambers.

[C] Deviation 3: Test concentrations were not measured for each test substance renewal and 

some test concentration measurements were performed on single replicate samples. Specifically, 

test concentrations were not measured on “old” samples (taken just before each renewal with 

fresh medium) collected on days 2 or 4 for the lowest test concentration (i.e., nominal 

concentration of 1000 μg/L). In addition, for the same concentration, only a single replicate was 

measured for “old” samples collected on days 1 and 3.

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 
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tests, the test substance should be measured in each replicate at the beginning and end of test 

and just before and after each renewal.”

Interpretation: Test concentration analyses should be performed on all replicates. Test fluid 

samples should be collected from each replicate at the beginning of the test, just before and after

each test substance renewal, and at the end of the test. All samples should be analyzed to 

determine test substance concentrations.

[D] Deviation 4: Details regarding the analytical measurements of test substance concentrations 

were missing from the study report, including the limit of detection of the analytical method and

chromatograms for the analytical measurements.

OPPTS 850.1075, pp. 10-11, Section (g) “Data and reporting”, Sub-section (2)(i) “Test 

report” states: “The test report must include the following:” and item (xvii) states:

“Methods used in analysis of test concentrations of the test chemical should be described. 

The accuracy of the method (i.e., detection limit and quantification limit) should be given.”

OPPTS 850.1075, p. 2, Section (c) “Principle of the test”, Sub-section (3) “Information 

on the test substance” states: “... A reliable analytical method for quantification of test 

substance concentrations must be available.”

OPPTS 850.1075, p. 2, Section (d) “Validity of the test”, Sub-section (4) states: “... 

[T]here must be evidence that test concentrations remained at least 80 percent of the nominal 

concentrations throughout the test or that mean measured concentrations are an accurate 

representation of exposure levels maintained throughout the test period.”

ASTM E729-96, p. 18, Section 15 “Report”, Sub-section 15.1 states: “The record of the 

results of an acceptable acute toxicity test should include the following information either 

directly or by reference to available documents:” and item 15.1.9 states: “Methods used for, 

and results (with standard deviations or confidence limits) of, chemical analyses of water 

quality and concentrations(s) of test material, impurities, and reaction and degradation 

products, including validation studies and reagent blanks.”

ASTM E729-96, p. 16, Section 12 “Analytical Methodology”, Sub-section 12.2 states:

“Chemical and physical data should be obtained using appropriate ASTM standards whenever 

possible. For those measurements for which ASTM standards do not exist or are not sensitive 

enough, methods should be obtained from other reliable sources.”

Interpretation: The analytical method used for measurement of test substance concentrations 

should be fully described. The results of validation studies of the analytical method should be 

provided. The limit of detection should be provided. The analytical results, including all data 

that are generated (e.g., chromatograms) and used to determine test substance concentrations in 

samples taken from test chambers over the course of the study, should be fully described and 

documented. The test report must provide evidence that the analytical method is reliable and 

accurate. The lack of complete details precludes an evaluation of whether or not the analytical 

method adheres to acceptable standards, such as ASTM. 

REFERENCE

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

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

16, 2014.
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Study 2: Acute Toxicity to Daphnia (Naddy 2012)

Relevant Guidelines: Study was conducted according to OPPTS 850.1010 and ASTM E729-96

[A] Deviation 1: This study failed to identify an EC50 because mortality and immobility at the 

highest test concentration were less than 50% in the definitive test. The study only determined 

that the EC50 was greater than 943 μg/L (i.e., 0.943 mg/L), which is considerably less than the 

reported water solubility limit of the test substance (2130 μg/L at 20°C).

OPPTS 850.1010, p. 2, Section (d) “Test procedures”, Sub-section (3) “Definitive test”, 

item (i) states: “The purpose of the definitive test is to determine the concentration-response 

curves and the 24- and 48-h EC50 values.”

OPPTS 850.1010, p. 3, Section (d) “Test procedures”, Sub-section (3) “Definitive test”, 

item (ii) states: “The concentration ranges should be selected to determine the concentrationresponse curves and EC50 values at 24 and 48 h.”

OPPTS 850.1010, p. 3, Section (d) “Test procedures”, Sub-section (3) “Definitive test”, 

item (v) states: “... Concentration-response curves and 24-h and 48-h EC50 values for 

immobilization should be determined along with their 95 percent confidence limits.”

OPPTS 850.1010, p. 8, Section (f) “Reporting” states: “... In addition to the reporting 

requirements prescribed in 40 CFR Part 792 – Good Laboratory Practice Standards, the 

reporting of test data should include the following:”; and item (10) states: “(10) The 24- and 

48-h EC50 values and their respective 95 percent confidence limits using the mean measured 

test chemical concentration, and the methods used to calculate both the EC50 values and their 

confidence limits.”

ASTM E729-96, p. 9, Section 9 “Test Material”, Sub-section 9.3 “Test 

Concentration(s)”, item 9.3.2 states: “In some (usually regulatory) situations, it is necessary

only to determine (1) whether a specific concentration of test material is acutely toxic to the 

test species, or (2) whether the LC50, EC50, or IC50 is above or below a specific concentration. 

For example, the specific concentration might be the concentration occurring in a surface 

water, the concentration resulting from the direct application of the material to a body of 

water, or the solubility limit of the material in water. When there is interest only in a specific 

concentration, it is often necessary only to test that concentration (see 11.1.1.2) and it is not 

necessary to actually determine the LC50, EC50, or IC50.”

Interpretation: A primary objective of an acute toxicity to Daphnia test is to determine a 

discrete EC50 value. If a discrete EC50 value is not determined because insufficient (< 50%) 

immobilization occurs at the highest concentration tested and this concentration is below the 

aqueous solubility or dispersibility limit, the test is not considered acceptable.

[B] Deviation 2: Measured concentrations increased from the beginning to the end of the 24-

hour renewal intervals, but the study report did not provide a valid explanation. 

OPPTS 850.1010, p. 4, Section (d) “Test procedures”, Sub-section (4) “Analytical 

measurements”, item (i) “Test chemical” states: “... The analytical method used to 

measure the amount of test chemical in a sample should be validated before beginning the test 

by appropriate laboratory practices.”

OPPTS 850.1010, p. 8, Section (f) “Reporting” states: “... In addition to the reporting 

requirements prescribed in 40 CFR Part 792 – Good Laboratory Practice Standards, the 

reporting of test data should include the following:”; and item (11) states: “All chemical 
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analyses of water quality and test chemical concentrations, including methods, method 

validations, and reagent blanks.”; and item (13) states: “Any deviation from this test

guideline and anything unusual about the test, e.g. diluter failure, temperature fluctuations, 

etc.”

ASTM E729-96, p. 18, Section 15 “Report”, Sub-section 15.1 states: “The record of the 

results of an acceptable acute toxicity test should include the following information either 

directly or by reference to available documents:” and item 15.1.3 states: “Anything unusual 

about the test, any deviation from these procedures, and any other relevant information.”

ASTM E729-96, p. 16, Section 11 “Procedure”, Sub-section 11.9.3 “Test Material”, item 

11.9.3.1 states: “... If the test material is not uniformly dispersed in the test chamber in static 

and renewal tests, the whole volume of solution in the test chamber should be (1) used as the 

sample or (2) treated appropriately (for example, by adding acid, base, or surfactant and 

mixing thoroughly) to uniformly distribute the test material before a sample is taken.”

Interpretation: Test concentrations should be determined with a validated analytical method. 

The report should address any deviations from the protocol or unusual occurrences that could 

impact the results. If the test substance is not homogeneously distributed in the fluid in the test 

chambers, additional measures should be taken to ensure that samples collected for analysis are 

representative of actual mean concentrations of test substance in the test chambers. 

[C] Deviation 3: Variation in test item concentrations among the replicates for “old” samples 

(taken just before each renewal with fresh medium) collected on day 2 was greater than 20 

percent.

OPPTS 850.1010, p. 3, Section (d) “Test procedures”, Sub-section (3) “Definitive test”, 

item (viii) states: “... Among replicate test chambers of a treatment concentration, the 

measured concentration of the test chemical should not vary more than ± 20 percent.”

Interpretation: Measured concentrations of samples taken from replicate test vessels of a given 

test item concentration should not vary by more than 20 percent from each other.

[D] Deviation 4: Details regarding the analytical measurements of test substance concentrations 

were missing from the study report, including the limit of detection of the analytical method and

chromatograms for the analytical measurements. 

OPPTS 850.1010, p. 4, Section (d) “Test procedures”, Sub-section (4) “Analytical 

measurements”, item (i) “Test chemical” states: “... The analytical method used to 

measure the amount of test chemical in a sample should be validated before beginning the test 

by appropriate laboratory practices.”

OPPTS 850.1010, p. 8, Section (f) “Reporting” states: “... In addition to the reporting 

requirements prescribed in 40 CFR Part 792 – Good Laboratory Practice Standards, the 

reporting of test data should include the following:” and item (11) states: “All chemical 

analyses of water quality and test chemical concentrations, including methods, method 

validations, and reagent blanks.”

ASTM E729-96, p. 18, Section 15 “Report”, Sub-section 15.1 states: “The record of the 

results of an acceptable acute toxicity test should include the following information either 

directly or by reference to available documents:” and item 15.1.9 states: “Methods used for, 

and results (with standard deviations or confidence limits) of, chemical analyses of water 
Draft Addendum to HPV2 Test Rule Data Adequacy Review (April 2014) CASRN 78-11-5

A-7

quality and concentrations(s) of test material, impurities, and reaction and degradation 

products, including validation studies and reagent blanks.”

ASTM E729-96, p. 16, Section 12 “Analytical Methodology”, Sub-section 12.2 states:

“Chemical and physical data should be obtained using appropriate ASTM standards whenever 

possible. For those measurements for which ASTM standards do not exist or are not sensitive 

enough, methods should be obtained from other reliable sources.”

Interpretation: The analytical method used for measurement of test substance concentrations 

should be fully described. The results of validation studies of the analytical method, including 

the limit of detection, should be provided. The analytical results, including all data that are 

generated (e.g., chromatograms) and used to determine test substance concentrations in samples 

taken from test chambers over the course of the study, should be fully described and 

documented. The measurement of test substance concentrations should be conducted using a 

validated analytical method. The lack of complete details regarding the analytical method 

precludes a full evaluation of the validity of the analytical method, including whether or not it 

adhered to acceptable standards, such as ASTM.

REFERENCE

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

16, 2014.