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 Work Assignment Manager 

March 5, 2010 
HPV1 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 22202 
Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 112-52-7

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. 

1 
Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 112-52-7

TOXICITY TO PLANTS (ALGAE) 

Title: 1-Chlorododecane: A 96-Hour Toxicity Test with the Freshwater Alga 

(Pseudokirchneriella 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 colorless to pale yellow liquid, 

stored at room temperature and was stable for the duration of testing. Batch #N6227945, Purity 

97.42%. 

METHOD

Method/guideline followed: U.S. EPA Health Effects Test Guideline OPPTS 850.5400, ASTM 

Standard E 1218-97a; EU Directive 92/96/EEC, Method 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, Maryland. Algal cells used in this test were 

obtained from Wildlife International, Ltd. cultures that had been actively growing in culture 

medium for at least two weeks prior to test initiation. 

Analytical monitoring: Yes 

Exposure duration: 96 Hours

Concentrations tested: 

Nominal: 0 (negative control), 0 (solvent 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 control (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 exploratory range finding toxicity test. 

2 
Draft HPV1 Test Rule – Data Adequacy – Toxicity 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 regression. 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 tests, 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 pooled control using ANOVA and Dunnett’s test 

(p=0.05). The results of the statistical analyses, as well as an evaluation of the concentrationresponse pattern, were used to determine the NOAEC relative to each parameter at 72 and 96 

hours. 

Analytical Methods: 

The method used for the analysis of 1-chlorododecane in freshwater algal medium samples was 

based upon methodology developed by Wildlife International, Ltd. The analytical method 

consisted of extracting the freshwater algal medium samples with ethyl acetate and analyzing by 

gas chromatography with mass selective detection. 

The method limit of quantitation (LOQ) for these 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 possible interferences. No interferences were observed at or above 

the LOQ during the sample analyses. 

RESULTS

Endpoint value(s): 

Cell Density: 

72-hour EC50: 0.029 mg a.i. /L 

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

96-hour EC50: > 0.034 mg a.i. /L 

96-hour NOAEC: 0.0076 mg a.i. /L 

Growth Rate: 

72-hour ErC50: > 0.034 mg a.i. /L 

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

96-hour ErC50: > 0.034 mg a.i./L 

96-hour NOAEC: 0.012 mg a.i./L 

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

Area Under the Growth Curve: 

72-hour EbC50: 0.027 mg a.i. /L 

72-h NOAEC: 0.0076 mg a.i. /L 

96-hour EbC50: 0.036 mg a.i. /L 

96-hour NOAEC: 0.0076 mg a.i. /L 

Results remarks: 

At test initiation, algal cells appeared normal. After 96-hours of exposure there were no 

noticeable changes in cell morphology at any of the concentrations tested. 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 growth rate in the control replicates during the 

exposure period did not exceed 7%, meeting the validity criteria set forth in the guidelines and 

protocol. No significant differences between the negative control and solvent control groups 

were found (p>0.05); therefore, control data were pooled for comparison with the treatment 

groups. 

Statistical Results: 

After 72 hours of exposure, statistically significant (p<0.05) reductions in area 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 control values. After 96 hours of exposure, there 

were statistically significant (p<0.05) reductions in cell density, area under the 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) reductions in cell density and area 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 the 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. The 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. 

4 
Draft HPV1 Test Rule – Data Adequacy – Toxicity 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; Gallagher, S.P., Kendall, T.Z, and Krueger, H.O. 2008. 1-

Chlorododecane: A 96-Hour Toxicity Test with the Freshwater Alga (Pseudokirchneriella 

subcapitata). Wildlife International Ltd. for Lonza Inc. Study No. 289A-165 (unpublished 

report). 

5 
Review 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 Toxicity 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 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 [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): 

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).