SRC TR-10-058 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

Methyl Heptanone, CAS No. 409-02-9 

Toxicity to Plants (Algae) 

Prepared by: 

Kendra Moran, Ashlee Aldridge, 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 

Methyl Heptanone, CAS No. 409-02-9 

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. 409-02-9

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. 

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Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 409-02-9

TOXICITY TO PLANTS (ALGAE) 

Title: Algae Growth Inhibition Test 

This study is: Adequate Not Adequate 

The submitter uses OECD TG 201 (1984) instead of the more recent OECD TG 201 (2006). 

TEST SUBSTANCE 

 

Identity: Methyl Heptanone, CAS No. 409-02-9 

Remarks: The test substance, Baton Rouge MEK Heavy Ketone Heart Cut C8 Ketone Fraction, 

was characterized as a complex mixture. 

METHOD

Method/guideline followed: OECD TG 201 (1984) 

Test type: Static

GLP compliant?: Yes

Year study was performed: 2007

Test species Pseudokirchneriella subcapitata. The algae were cultured in-house from 5 day old 

stock cultures in log phase growth. 

Analytical monitoring: Yes 

Exposure duration: 72-hrs

Concentrations tested: 0 (control), 0.8, 1.6, 4.1, 8.8, and 20 mg/L test substance water 

accomadated fraction (WAF), which corresponded to mean measured concentrations of 0 

(control), 0.715, 1.42, 3.28, 7.71, and 12.9 mg/L.

Test method/conditions remarks: Individual Water Accommodated Fractions (WAFs) were 

prepared for each treatment. The test substance was added to 4.0 L of algal nutrient medium 

augmented with sodium bicarbonate in glass aspirator bottles (capacity 4.5 L). The solutions 

were mixed for 24 hours using an 9.5% vortex (of the static liquid depth). The test solutions 

were removed through the outlet at the bottom of each mixing vessel into nine replicates of 

approximately 140 mL in 125 mL Erlenmeyer flasks (no headspace) containing two 14-mm glass 

spheres to facilitate mixing. The test chambers were inoculated with algae (1.0 x 104 cells/mL) 

and were closed with ground glass stoppers. Three replicates were sacrificed daily for cell 

density determination. The test chambers were placed on shaker tables (101 rpm) to keep the 

algae in suspension. The test was performed under static conditions with no aeration. 

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Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 409-02-9

 

 Test temperature ranged from 23.6 to 23.9°C. Cultures were exposed to continuous light with 

an intensity of 7700 to 8400 Lux. The pH was 7.6 in the test solutions at test initiation and 

ranged from 8.6 to 8.7 at test termination. 

Due to the complex nature and limited water solubility of the test substance, the following 

exceptions to the guideline apply for this study: 1) The concentration of the test substance in 

solution was not determined prior to us; 2) Test substance analysis was performed on samples of 

the WAFs at the start of the test (day 0) and at termination (day 4); and 3) It was appropriate to 

prepare individual treatment solutions by adding the test substance to dilution water and 

removing the WAF of each mixture for testing than to prepare dilutions of a stock solution. 

None of the above exceptions are believed to have affected the outcome, integrity, or quality of 

the study. 

RESULTS

Endpoint value(s): Based on mean measured concentrations, the 72-hour ErC50 was 16.0 mg/L 

and the 72-hour EbC50 was 8.00 mg/L. The 72-hour NOEC for growth rate was 3.28 mg/L and 

for biomass (area under the curve) was 0.715 mg/L. 

Based on WAF loading rates, the 72-hour ErL50 was 24.7 mg/L and the 72-hour EbL50 was 11.3 

mg/L. The 72-hour NOEL for growth rate was 4.1 mg/L and for biomass (area under the curve) 

was 0.8 mg/L. 

Results remarks: Controls responded adequately based on an increase in cell density of 

approximately 54x over the 72 hour exposure period. 

CONCLUSIONS

After algae were exposed to a water accommodated fraction (WAF) prepared from Baton Rouge 

MEK Heavy Ketone Heart Cut - C8 Ketone Fraction for 72 hours, the EC50 was 16 mg/L based 

on mean measured concentrations and the EL50 was 24.7 mg/L based on WAF loading rates for 

growth rate, and the EC50 was 8 mg/L based on mean measured concentrations and the EL50 was 

11.3 mg/L based on WAF loading rates for biomass. 

STUDY RELIABILITY

[2] Reliable with restrictions; the study used a 72-hour duration instead of the ASTM E 1218 

specified 96-hour duration. 

REFERENCE

ExxonMobil Biomedical Sciences, Inc. 2008. ALGA, GROWTH INHIBITION TEST. Study # 

0700867. 

3 
Review of TSCA Section 4 Final Study Report 

CITB Project Manager: Catherine Roman 

Chemical Name: Methyl Heptanone 

CAS Registry Number: 409-02-9 

Report Title: Alga Growth Inhibition Test 

Test Sponsor: Exxon Mobil Chemical Company 

Testing Lab: Exxon Mobil Biomedical Sciences, Inc. 

Test Standard: ASTM E 1218 

Date Study Received by EPA: 6/23/09 (date posted to http://www.regulations.gov) 

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. 

 [A] 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. 

 

 [A] 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. 

[D] 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. [ ] 

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

 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 [x] 

 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): 

The study used a 72-hour duration instead of the ASTM E 1218 specified 96-hour duration. Initial inoculum (1 x 104

cells/mL) was less then specified in ASTM E 1218 (2 x 104

 cells/mL). Algae nutrient medium was not as specified 

in ASTM E 1218. 

Reviewer Name/Title/Branch/Division/Office: Kendra Moran/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).