SRC TR-10-063

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

1,3-Propanediol, 2,2-bis[(nitr ooxy)methyl]-, dinitrate (e ster), 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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

PREPARATION OF THE DAT A ADEQUACY ASSESSMENT

This document contains the review of a toxi city 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 guide s, the study was reviewed for adequacy and evaluated for compliance with the test guideline. (T he applicable test guid eline 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 assi gned a Klimisch reliability code. If the study was determined to be inadequate, a basis for th at conclusion is supplie d. For studies already assigned Klimisch codes, an explanation is prov ided if the reviewer’s assessment of data adequacy differed from the pre-assigned Klimisch code.

A detailed evaluation of the test report a nd study conduct is provided at the end of the summary document in a section entitled “Revie w 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 Procedur es, Good Laboratory Prac tice Standards, and Quality Assurance procedures, as well as the use of appropriate controls and test substances. Any deficiencies in the descri ptions of the study method, test system, animals studied, physical conditions, dosage information, indi vidual 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.

1Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

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 tetr anitrate (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 ( Pseudokirchnerie lla subcapitata , formerly Selenastrum capricornutum ); source: 4-day old in-hous e stock cultures; starter cu lture 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 (a s 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 ster ile, 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. Additi onally, the medium was sterilized by filtration through a pre-washed 0.45 μm cellulose membrane filter prior to mixing test solutions.

2Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

Macro- and micro-nutrients were added to the 100% test solution prio r to preparing test concentrations. The test substance, with adde d nutrients, was assumed to be 100% for purposes of test concentration preparation. Test concen trations 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-h our static test design; test solutions

were not renewed. S. capricornutum were exposed to four con centrations of PETN (using a dilution factor of 10), and a dilu tion water control. Nominal te st concentrations were 100, 10, 1, 0.1, and 0% PETN. Test solutions were prepar ed on test Day 0 immedi ately prior to test

initiation.

Test chambers were 250-mL (nominal volume) gl ass flasks containing ap proximately 100 mL of test solution and capped with a plas tic cup with three small holes. Test chambers were held in an environmental chamber in a five by five pattern. Test chambers were plac ed within the pattern according to a computer-generated random design.

The test chambers were housed in an envir onmental 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 oscillat ed continuously at approximately 78 rpm on a mechanical shaker table to keep the algal cells in suspensi on. 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 prio r to test initiation. The cell density of the

inoculum was 1.14 x 10 6 cells/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 10 4 cells/mL. This initial cell density was verified in three control blanks that were preserved with Lugol's solutio n at the rate of 1% of the bl ank volume within 2 hours of test initiation. Five replicates were tested per treat ment; one replicate was used to monitor pH during

the test and was discarde d at test termination.

On the day of test initiation, temperature, pH, and conductivity were measured and recorded for each test treatment. Hardness (mg/L CaCO 3 ), alkalinity (mg/L CaCO 3 ), 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 soluti ons destined for cell density dete rmination were preserved with Lugol's solution at a rate of 1% of the final vol ume within 2 hours of test termination, and after collection of analytical samples. Algal cell densit y in the test solutions was later determined by direct microscopic counts (using a hemocy tometer and compound light microscope) and

recorded.

3Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

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 determinati on, 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 memb rane to remove algal cells. Both samples were collected for each treatment (one with ce lls and one without) by pouring the composite test solution into 250-mL clear glas s bottles, wrapped in aluminum foil. The samples were refrigerated until shipped (on ice) to TestAmer ica, an independent laboratory, located in Knoxville, Tennessee. Method 8330 was used fo llowing USEPA (1986) guidelines to analyze

for nitroaromatics and nitramines by HPLC. Analytical data fr om TestAmerica were reviewed by ENSR. All data were determin ed to be valid and usable.

Statistical Analysis Because cell density in any te st concentration (including 100%) was not reduced by more than 8% from the control, a 96-hour EC 50 or IC 50 value (experimentally derive d 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 EC 50 > 2850 μg/L PETN

Results remarks: At the time of test initiation, water quality measurements in dilution water were: hardness = 18 mg/L CaCO 3 , alkalinity = 10 mg/L CaCO 3 , 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 CaCO 3 , alkalinity = 11 mg/L CaCO 3 , 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 initiatio n) = 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 10 5 cells/mL for the control, 3.2, 30, 310, and 2850 μg/L concentrations, respectively. The

EC 50 value, based on cell density and measured PETN concentrations, was > 2850 μg/L; cell density in any test concentrati on did not decrease more than 8% relative to the control.

CONCLUSIONS

The purpose of this study was to determine th e short-term chronic toxicity of PETN to S. capricornutum under static test conditions. The bi ological endpoint for this study was cell density (cells/ml). A 96-hour EC 50 was determined based on mean measured PETN concentration (μg/L) and cell density. The EC 50 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.

4Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 78-11-5

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.

5Review 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 Un icellular 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) inspect ions, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures 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 summar y 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 ri sk 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 guid eline states that each test chamber shoul d be inoculated at the rate of 2 x 10 4 cells/mL, the initial cell density in this study was only 1.14 x 10 4 cells/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 dens ities in test article-treated cultures were similar to controls, the EC 50 value exceeded the highest co ncentration tested; therefore, no statistics were necessary.

Reviewer Name/Title/Bran ch/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 me t, 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).