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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy – T oxicity to Plants (Algae) CAS No. 409-02-9

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. 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) instea d 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 H eavy 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?: Ye s

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 Accommoda ted Fractions (WAFs) were prepared for each treatment. The test substan ce was added to 4.0 L of algal nutrient medium augmented with sodium bicarbonate in glass as pirator bottles (capacity 4.5 L). The solutions were mixed for 24 hours using an 9.5% vortex (of th e 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 flas ks (no headspace) containing two 14-mm glass spheres to facilitate mixing. Th e test chambers were inoculated with algae (1.0 x 104 cells/mL) and were closed with ground glass stoppers. Thr ee 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 performe d under static conditi ons with no aeration.

2Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 409-02-9

Test temperature ranged from 23.6 to 23.9°C. Cu ltures 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 pe rformed 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 so lutions by adding the test su bstance 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, in tegrity, or quality of the study.

RESULTS

Endpoint value(s): Based on mean measured concentrations, the 72-hour E

r C50 was 16.0 mg/L

and the 72-hour E b C50 was 8.00 mg/L. The 72-hour NOEC fo r 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 E

r L50 was 24.7 mg/L and the 72-hour E b L50 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 base d on an increase in cell density of approximately 54x over the 72 hour exposure period.

CONCLUSIONS After algae were exposed to a water accommodate d fraction (WAF) prepared from Baton Rouge MEK Heavy Ketone Heart Cut - C8 Ke tone Fraction for 72 hours, the EC 50 was 16 mg/L based

on mean measured concentrations and the EL 50 was 24.7 mg/L based on WAF loading rates for growth rate, and the EC 50 was 8 mg/L based on mean measured concentrations and the EL 50 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.

3Review 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) 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 di fferent 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 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. [ ] 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 10 4

cells/mL) was less then specified in ASTM E 1218 (2 x 10 4 cells/mL). Algae nutrient medium was not as specified

in ASTM E 1218.

Reviewer Name/Title/Bran ch/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 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).