SRC TR-10-063

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester), CAS No. 78-11-5

Toxicity to Plants (Algae)

Prepared by:

Ashlee Aldridge, Teresa Manyin, and Joan D. Garey

Environmental Science Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.004

Submitted to:

OPPT/RAD/HPVCB (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

March 5, 2010
HPV1 Test Rule – Data Adequacy Assessment

1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester), CAS No. 78-11-5

Toxicity to Plants (Algae)

March 2010

Submitted to:

High Production Volume Chemicals Branch 

Risk Assessment Division 

Office of Pollution Prevention and Toxics 

Environmental Protection Agency 

WAM: Meena Sonawane

Alternate WAM: David Brooks

1200 Pennsylvania Avenue, NW 

Washington, DC 20460-0001

Prepared By:

SRC, Inc. 

Environmental Science Center 

2451 Crystal Drive, Suite 804 

Arlington, VA 22202
Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

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PREPARATION OF THE DATA ADEQUACY ASSESSMENT

This document contains the review of a toxicity study conducted in response to the first 

HPV TSCA Section 4 Test Rule (71 FR 13708, March 16, 2006). Using the applicable test 

guideline and professional judgment as guides, the study was reviewed for adequacy and 

evaluated for compliance with the test guideline. (The applicable test guideline is not always the 

test guideline required by the test rule, because some test sponsors requested and received 

approval from EPA to use a different guideline.) The study was summarized according to a 

standard format, assessed for adequacy, and assigned a Klimisch reliability code. If the study

was determined to be inadequate, a basis for that conclusion is supplied. For studies already 

assigned Klimisch codes, an explanation is provided if the reviewer’s assessment of data 

adequacy differed from the pre-assigned Klimisch code. 

A detailed evaluation of the test report and study conduct is provided at the end of the 

summary document in a section entitled “Review of TSCA Section 4 Final Study Report”. Any 

deviations from the applicable test guideline are described. The study is also evaluated for 

compliance with Standard Operating Procedures, Good Laboratory Practice Standards, and 

Quality Assurance procedures, as well as the use of appropriate controls and test substances. 

Any deficiencies in the descriptions of the study method, test system, animals studied, physical 

conditions, dosage information, individual animal data, calculations, and statistical analyses are 

described, and internal inconsistencies within the study report are identified. An overall 

assessment is provided for each of the following: study conduct, data reliability, and data utility.
Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

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TOXICITY TO PLANTS (ALGAE)

Title: Toxicity of Pentaerythritol tetranitrate (PETN) to the Unicellular Green Alga (Selenastrum 

capricornutum) under Static Test Conditions

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Pentaerythritol tetranitrate (1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate 

(ester); PETN), CAS No. 78-11-5

Remarks: Test substance was received as a solution of PETN (> 99% purity) dissolved in water 

(approximately 2100 μg/L); clear, colorless liquid; specific gravity: 1.77 g/cc; received from the 

United States Army Center for Health Promotion and Preventative Medicine

METHOD

Method/guideline followed: ASTM E 1218-04 (ASTM 2006) and where applicable OPPTS

850.5400 (USEPA 1996)

Test type: Toxicity to Algae

GLP compliant?: Yes

Year study was performed: 2008

Test species: Unicellular green algae (Pseudokirchneriella subcapitata, formerly Selenastrum 

capricornutum); source: 4-day old in-house stock cultures; starter culture obtained from Carolina 

Biological Supply Company

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: Nominal: 0, 0.1, 1.0, 10, and 100% PETN (as received), equivalent to 0, 

2, 20, 200 and 2000 μg/L; Mean measured: 1.4, 3.2, 30, 310 and 2850 μg/L

Test method/conditions remarks:

The dilution/control water used in this study (algal medium batch #121807) was ASTM algal 

growth medium with EDTA, prepared with sterile, deionized (Milli-Q®) water (ASTM type I) 

according to ASTM guidelines (ASTM 2006). Milli-Q® water generated at the testing lab is 

periodically analyzed for contaminants. Additionally, the medium was sterilized by filtration 

through a pre-washed 0.45 μm cellulose membrane filter prior to mixing test solutions.
Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

3

Macro- and micro-nutrients were added to the 100% test solution prior to preparing test 

concentrations. The test substance, with added nutrients, was assumed to be 100% for purposes 

of test concentration preparation. Test concentrations are expressed as percent of PETN as 

received; statistical endpoints are expressed as μg/L PETN, per the analytical chemistry data.

S. capricornutum were exposed to PETN according to a 96-hour static test design; test solutions

were not renewed. S. capricornutum were exposed to four concentrations of PETN (using a

dilution factor of 10), and a dilution water control. Nominal test concentrations were 100, 10, 1,

0.1, and 0% PETN. Test solutions were prepared on test Day 0 immediately prior to test 

initiation.

Test chambers were 250-mL (nominal volume) glass flasks containing approximately 100 mL of

test solution and capped with a plastic cup with three small holes. Test chambers were held in an 

environmental chamber in a five by five pattern. Test chambers were placed within the pattern 

according to a computer-generated random design.

The test chambers were housed in an environmental chamber under continuous illumination at

400 ± 10% ft-c (4.3 K lux), which was provided by a combination of incandescent and coolwhite fluorescent lamps to provide light output in the spectral range of 400-700 nm. Light 

intensity was monitored at the beginning of the test at the level of the test solutions and averaged 

398 ft-c. Flasks were oscillated continuously at approximately 78 rpm on a mechanical shaker 

table to keep the algal cells in suspension. Test temperature was monitored using a 

minimum/maximum thermometer in the environmental chamber and was 25 °C throughout the 

test duration.

The inoculum was prepared approximately 3 hours prior to test initiation. The cell density of the

inoculum was 1.14 x 106cells/mL. The inoculum was added to the test solution at a rate of 1 mL 

of inoculum per 100 mL of test solution. The initial cell density in the test chambers was 

nominally 1.14 x 104cells/mL. This initial cell density was verified in three control blanks that 

were preserved with Lugol's solution at the rate of 1% of the blank volume within 2 hours of test 

initiation. Five replicates were tested per treatment; one replicate was used to monitor pH during 

the test and was discarded at test termination.

On the day of test initiation, temperature, pH, and conductivity were measured and recorded for 

each test treatment. Hardness (mg/L CaCO3), alkalinity (mg/L CaCO3), total ammonia (mg/L as 

N), and total residual chlorine (mg/L) were also measured in the control and 100% PETN 

treatments at test initiation. Temperature and pH were measured in each concentration daily and 

at test termination. Temperature within the environmental chamber was measured daily using a 

digital thermometer, and recorded.

At test termination, test solutions destined for cell density determination were preserved with 

Lugol's solution at a rate of 1% of the final volume within 2 hours of test termination, and after 

collection of analytical samples. Algal cell density in the test solutions was later determined by 

direct microscopic counts (using a hemocytometer and compound light microscope) and 

recorded.
Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

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Samples for PETN analysis were collected at test initiation, prior to distributing the test solutions

to the test chambers, from each freshly prepared test treatment and the dilution water. At test

termination, samples of the old test solutions were collected after aliquots were removed for

algal growth-response determination, and the replicates for each treatment were pooled into one 

sample. The pooled sample was divided into two equal volumes. One of these samples was 

filtered through a pre-washed 0.45 μm cellulose membrane to remove algal cells. Both samples 

were collected for each treatment (one with cells and one without) by pouring the composite test 

solution into 250-mL clear glass bottles, wrapped in aluminum foil. The samples were 

refrigerated until shipped (on ice) to TestAmerica, an independent laboratory, located in

Knoxville, Tennessee. Method 8330 was used following USEPA (1986) guidelines to analyze 

for nitroaromatics and nitramines by HPLC. Analytical data from TestAmerica were reviewed 

by ENSR. All data were determined to be valid and usable.

Statistical Analysis

Because cell density in any test concentration (including 100%) was not reduced by more than

8% from the control, a 96-hour EC50 or IC50 value (experimentally derived concentration of a test

substance in dilution water that is calculated to affect 50% of a test population over 96 hours)

could not be statistically derived.

RESULTS

Endpoint value(s): 96 hr EC50 > 2850 μg/L PETN

Results remarks:

At the time of test initiation, water quality measurements in dilution water were: hardness = 18 

mg/L CaCO3, alkalinity = 10 mg/L CaCO3, pH = 7.5, conductivity = 95 μS/cm, total ammonia < 

1.0 mg N/L, and total residual chlorine < 0.02 mg/L. Water quality measurements of 100% 

PETN (with algal nutrients) were: hardness = 16 mg/L CaCO3, alkalinity = 11 mg/L CaCO3, pH

= 7.7, conductivity = 82 μS/cm, total ammonia < 1.0 mg N/L, and total residual chlorine < 0.03 

mg/L. The physical test conditions monitored during the test were: pH = 7.5-9.4, conductivity

(measured only at test initiation) = 82-95 μS/cm, and temperature = 24-26 °C. The conditions 

were within acceptable ranges for the survival of S. capricornutum and were in compliance with 

the protocol and ASTM requirements.

The average cell densities after 96 hours of exposure were 25.63, 29.42, 31.71, 26.39, and 23.81 

x 105cells/mL for the control, 3.2, 30, 310, and 2850 μg/L concentrations, respectively. The 

EC50 value, based on cell density and measured PETN concentrations, was > 2850 μg/L; cell 

density in any test concentration did not decrease more than 8% relative to the control.

CONCLUSIONS

The purpose of this study was to determine the short-term chronic toxicity of PETN to S.

capricornutum under static test conditions. The biological endpoint for this study was cell 

density (cells/ml). A 96-hour EC50 was determined based on mean measured PETN 

concentration (μg/L) and cell density. The EC50 was > 2850 μg/L PETN. Because the highest 

concentration tested (2850 μg/L of PETN) did not result in a 50% or greater reduction in growth, 

a definitive test is not required.
Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

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STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

Pillard, D.A. 2008. Toxicity of Pentaerythritol tetranitrate (PETN) to the Unicellular Green 

Alga (Selenastrum capricornutum) under Static Test Conditions. ENSR Project ID 08503-131-

073-002, Study #08503-131-073-002. Study completed 13 March 2008.
Review of TSCA Section 4 Final Study Report 

CITB Project Manager: Catherine Roman

Chemical Name: 1,3-Propanediol, 2,2-bis[(nitrooxy)methyl]-, dinitrate (ester)

CAS Registry Number: 78-11-5

Report Title: Toxicity of Pentaerythritol tetranitrate (PETN) to the Unicellular Green Alga (Selenastrum 

capricornutum) under Static Test Conditions 

Test Sponsor: Dyno Nobel Inc., Salt Lake City, UT

Testing Lab: ENSR Fort Collins Environmental Toxicology Laboratory, Fort Collins, CO

Test Standard: ASTM E 1218

Date Study Received by EPA: N/A, posted to http://www.regulations.gov on 5/19/08

Date Study Due to EPA: 5/17/07

Special Instructions (e.g., regarding approved protocol modifications, etc.):

Part I. Test Report and Study Conduct

Enter one of the following codes into each of the checkboxes 1 through 13: Agree [A], Disagree [D], Not 

Applicable [NA], or Cannot Determine [C], and add comment, below, as needed

[D] 1. The final study report indicates evidence of gross, unauthorized or undocumented deviation from 

the EPA test standard requirements, including “shall,” “must,” or “will” violations. List 

significant test standard deviations and “shall,” “must,” or “will” violations in comments below.

[D] 2. The final study report indicates evidence of deviations from the “should” statements in the study protocol 

that may have a bearing on the study conclusions. List these in comments below.

[D] 3. Approved modifications of test standard or testing protocol (CITB PM describe or attach) were not 

followed or are inadequately documented.

[D] 4. There is evidence that laboratory Standard Operating Procedures (SOPs) were inadequately followed.

[D] 5. The Statement of Quality Assurance (QA) inspections, their dates, and findings were missing or not signed. 

There is evidence that QA procedures may be inadequate.

[D] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed.

[D] 7. The test, control, or reference substances are different from that specified in the section 4 test rule or 

Enforceable Consent Agreement (ECA).

[D] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations 

from temperature, lighting, dosing) are apparent.

[D] 9. The final study report inadequately describes the study method.

[D] 10. The final study report inadequately describes the test system.
[D] 11. The final study report inadequately describes the dosage information.

[D] 12. The final study report does not accurately reflect the individual animal data (or that of other discrete 

observations pertinent to the type of study conducted), or individual data were not provided. 

[NA] 13. The final study report inadequately describes the calculations, interpolations, data transformations, 

statistical analyses, or modeling parameters used.

Part II. Conclusions

Place a check mark beside the summary statement(s) that apply to this review and add comments below as 

appropriate

Part A. To be filled out by Reviewer

Study Conduct 1. The study was well-conducted. [X]

2. The study was generally well-conducted, with some minor [ ]

 deficiencies (list in notes below).

3. The study conduct was significantly deficient or questionable, (give [ ]

 reasons in notes below).

Data Reliability 4. The study results are precise and accurate. [X]

5. Appropriate methods were used to determine [NA]

 statistical significance.

6. If repeated, the test results would be expected to be the [X]

 same.

Data Utility 7. The data are useful for risk assessment for the endpoint tested. [X]

8. The data are useful for some other purpose (e.g., dose-setting). [ ]

9. The data are not useful for any purpose (describe in notes below). [ ]

Reviewer Comments (continue on separate page, if needed): 

Although the test guideline states that each test chamber should be inoculated at the rate of 2 x 104cells/mL, the 

initial cell density in this study was only 1.14 x 104cells/mL. It is unlikely that this violation has bearing on the 

study conclusion. Cell density in control cultures increased by 225x during the 96-hour exposure period, which 

exceeds the minimum acceptable growth rate. Because cell densities in test article-treated cultures were similar to 

controls, the EC50 value exceeded the highest concentration tested; therefore, no statistics were necessary.

Reviewer Name/Title/Branch/Division/Office: Ashlee Aldridge/SRC, Inc.

Reviewer Signature: David Brooks/HPVCB/RAD/OPPTS

Part B. To be filled out by CITB PM

Tiered Testing Decision 1. The tiered testing trigger was met, additional testing is indicated. [ ] 

(when applicable) 2. The trigger for additional (tiered) testing was not met. [ ]

 

Study Acceptability 3. The study is acceptable. [ ]

4. The study may not be acceptable [ ]

 (provide rationale in memo to CITB chief).