SRC TR-10-051

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT

1-Chlorododecane, CAS No. 112-52-7

Toxicity to Plants (Algae)

Prepared by:

Ashlee Aldridge, Justine Von Runnen and Joan D. Garey

Environmental Science Center
SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.0.009.002

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-Chlorododecane, CAS No. 112-52-7

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. 112-52-7

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 – T oxicity to Plants (Algae) CAS No. 112-52-7

TOXICITY TO PLANTS (ALGAE)

Title: 1-Chlorododecane: A 96-Hour Toxicity Test with the Freshwater Alga ( Pseudokirchnerie lla subcapitata )

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: 1-Chlorododecane, CAS No. 112-52-7

Remarks: Identified as Barchlor 12s; The test substance was a colo rless to pale yellow liquid, stored at room temperature and was stable fo r the duration of testi ng. Batch #N6227945, Purity

97.42%.

METHOD

Method/guideline followed: U.S. EPA Health Effects Te st Guideline OPPTS 850.5400, ASTM Standard E 1218-97a; EU Directive 92/96/EEC, Me thod C.3 and OECD Test Guideline No. 201

Test type: Static

GLP compliant?: Yes

Year study was performed: 2008

Test species: Freshwater Alga ( Pseudokirchneriella subcapitata ). Original algal cultures were obtained from the University of Toronto Culture Collection and had been maintained in culture medium at Wildlife International, Ltd., Easton, Ma ryland. Algal cells used in this test were obtained from Wildlife International, Ltd. culture s that had been actively growing in culture

medium for at least two week s prior to test initiation.

Analytical monitoring: Yes

Exposure duration: 96 Hours

Concentrations tested: Nominal: 0 (negative control), 0 (solve nt control), 0.016, 0.031, 0.063, 0.13, 0.25, 0.50 mg a.i./L Measured: < LOQ, < LOQ, 0.0055, 0.0076, 0.012, 0.018, 0.024, 0.034 mg a.i./L

Test method/conditions remarks: The green alga ( Pseudokirchneriella subcapitata ) was exposed to six test concentrations, a negative control (culture medium ) and a solvent contro l (0.1 mL/L dimethyl formamide) for 96 hours. Three replicate test chambers in each control and each treatment group were maintained.

Nominal test concentrations were selected in consultation with the Sponsor and were based upon the results of an explorator y range finding toxicity test.

2Draft HPV1 Test Rule – Data Adequacy – T oxicity to Plants (Algae) CAS No. 112-52-7

At test initiation, an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of approximately 10,000 P. subcapitata cells/mL. Samples were collected from each replicate test chamber at approximately 24-hour intervals during the test to determine cell densities, which were subsequently used to calculate areas under the growth curve (biomass) and growth rates.

Statistical Methods: EC50 values were calculated by non-linear regres sion. The cell density, area under the growth curve (biomass) and growth rate data were evaluated for normality and homogeneity of variance (p=0.01) using the Shapiro-Wilk’s and Levene’s te sts, respectively. With the exception of the Day 4 growth rate data, the data were normal with homogeneous variances. A log transformation of the Day 4 data was unsuccessful in correcting the unequal error variances for the growth rate. Therefore, analysis of the Day 4 growth rate data was performed on the untransformed data. All treatment groups were compared to the pool ed control using ANO VA and Dunnett’s test (p=0.05). The results of the statis tical analyses, as well as an evaluation of the concentrationresponse pattern, were used to determine the N OAEC relative to each parameter at 72 and 96

hours.

Analytical Methods: The method used for the analysis of 1-chlorodod ecane in freshwater algal medium samples was based upon methodology developed by Wildlife Inte rnational, Ltd. The analytical method consisted of extracting the freshwater algal medi um samples with ethyl acetate and analyzing by gas chromatography with mass selective detection.

The method limit of quantitation (LOQ) for thes e analyses was defined as 0.00750 mg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.0150 mg a.i./L) and the dilution factor of the matrix blank samples (0.500). Two matrix blank samples were analyzed to determine possi ble interferences. No interferen ces were observe d at or above the LOQ during the sample analyses.

RESULTS

Endpoint value(s): Cell Density:

72-hour EC 50 : 0.029 mg a.i. /L 72-hour NOAEC: 0.0076 mg a.i. /L

96-hour EC 50 : > 0.034 mg a.i. /L 96-hour NOAEC: 0.0076 mg a.i. /L

Growth Rate:

72-hour E

r C 50 : > 0.034 mg a.i. /L 72-hour NOAEC: 0.0076 mg a.i. /L

96-hour E

r C 50 : > 0.034 mg a.i./L 96-hour NOAEC: 0.012 mg a.i./L

3Draft HPV1 Test Rule – Data Adequacy – T oxicity to Plants (Algae) CAS No. 112-52-7

Area Under the Growth Curve:

72-hour E b C 50 : 0.027 mg a.i. /L 72-h NOAEC: 0.0076 mg a.i. /L

96-hour E b C 50 : 0.036 mg a.i. /L 96-hour NOAEC: 0.0076 mg a.i. /L

Results remarks: At test initiation, algal cells appeared nor mal. After 96-hours of exposure there were no noticeable changes in cell morphology at any of the concentrations test ed. There was also no flocculation or aggregation of cells , or adherence of cells to test chambers noted in the controls

or treatment groups.

Temperatures ranged from 23.2 to 23.9°C and were within the 24 ± 2°C range established for the test. Measurements of pH were 7.6 in the test solutions at the beginning of the test period and ranged from 8.2 to 8.3 at test termination. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with P. subcapitata . The light intensity ranged from 3930 to 4500 lux, which was within the desired range of 4,300 ± 10% lux.

Control Response: Mean cell density in the control flasks increased by a factor greater than 16 within three days and the coefficient of variation of average specific gr owth rate in the contro l replicates during the exposure period did not exceed 7%, meeting the valid ity criteria set forth in the guidelines and protocol. No significant differe nces between the negative cont rol and solvent control groups were found (p>0.05); therefore, control data we re pooled for comparis on with the treatment

groups.

Statistical Results: After 72 hours of exposure, statistically significant ( p <0.05) reductions in ar ea under the growth curve, cell density and growth rate were observed in the 0.012, 0.018, 0.024 and 0.034 mg a.i./L treatment groups when compared to pooled cont rol values. After 96 hours of exposure, there were statistically significant (p <0.05) reductions in cel l density, area under th e growth curve and growth rate in the 0.018, 0.024 and 0.034 mg a.i./L treatment groups. There were also statistically significant (p<0.05) re ductions in cell density and ar ea under the growth curve in the 0.012 mg a.i./L treatment group.

CONCLUSIONS

Pseudokirchneriella subcapitata were exposed to six concentrations of 1-chlorododecane and evaluated for effects on cell density, area under the growth curve (biomass) and growth rate. Cell density and area under the growth curve were th e most sensitive endpoints, as defined by the lowest 96-hour NOAEC values (0.0076 mg a. i./L for both endpoints). The 96-hour NOAEC based on growth rate was 0.012 mg a.i./L. Th e 96-hour EC50 and ErC50 were both >0.034 mg a.i./L, the highest geometric mean measured concentration. The 96-hour EbC50 value was 0.036 mg a.i./L.

4Draft HPV1 Test Rule – Data Adequacy – T oxicity to Plants (Algae) CAS No. 112-52-7

The acute toxicity of the test substance to Pseudokirchneriella subcapitata has been adequately characterized (Lonza Inc.).

STUDY RELIABILITY

1 – Reliable without restrictions

REFERENCE

EPA-HQ-OPPT-2005-0033-0314.5; Gallaghe r, S.P., Kendall, T.Z, and Krueger, H.O. 2008. 1- Chlorododecane: A 96-Hour Toxicity Te st with the Freshwater Alga ( Pseudokirchneriella subcapitata ). Wildlife Internationa l Ltd. for Lonza Inc. Study No. 289A-165 (unpublished report).

5Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman

Chemical Name: 1-Chlorododecane

CAS Registry Number: 112-52-7

Report Title: 1-Chlorododecane: A 96-Hour Toxi city Test with the Freshwater Alga ( Pseudokirchneriella subcapitata )

Test Sponsor: Lonza, Inc.

Testing Lab: Wildlife International, Ltd.

Test Standard: ASTM E 1218

Date Study Received by EPA: September 19, 2008 Date Study Due to EPA: May 17, 2007

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.

[NA] 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 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 [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 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):

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).