SRC TR-10-053 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

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

Chronic Toxicity to Daphnia 

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 

Chronic Toxicity to Daphnia 

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 – Chronic Toxicity to Daphnia 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. 

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Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

CHRONIC TOXICITY TO DAPHNIA 

Title: A Flow-Through Life-Cycle Toxicity Study in 1-Chlorododecane with the Cladoceran 

(Daphnia magna) 

This study is: Adequate Not Adequate 

TEST SUBSTANCE 

 

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

Remarks: 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: The protocol was based on procedures outlined in the U.S. 

Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines, OPPTS 

Number 850.1300: Daphnid Chronic Toxicity Test; OECD Guideline 211: Daphnia magna 

Reproduction Test; and ASTM Standard E 1193-97: Standard Guide for Conducting Daphnia 

magna Life-Cycle Toxicity Tests 

Test type: Flow-Through

GLP compliant?: Yes

Year study was performed: 2008

Test species: Cladoceran (Daphnia magna), less than 24 hours old. Culture maintained by 

Wildlife International, Ltd., Easton, MD. 21601 

Analytical monitoring: Yes 

Exposure duration: 21 Days

Concentrations tested: 

Nominal: 0 (negative control), 0 (positive control), 6.3, 13, 25, 50 and 100 μg a.i./L 

Measured: < LOQ, < LOQ, 1.5, 2.7, 5.7, 7.2 and 17 μg a.i./L

Test method/conditions remarks: 

Daphnids were exposed to a geometric series of five test concentrations, a negative control 

(dilution water) and a solvent control (dimethyl formamide) for 21 days. Delivery of the test 

solutions to the test chambers was initiated six days prior to test initiation in order to achieve 

equilibrium of the test substance. Two replicate test chambers (25-L aquaria filled with 

approximately 22 L of test solution) were tested for each treatment and control. Each replicate 

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Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

contained two compartments (300-mL glass beakers) with five daphnids, resulting in a total of 

20 D. magna in each treatment and control group. 

At test initiation, neonate daphnids were impartially assigned to exposure chambers. Test 

chambers were indiscriminately positioned in a temperature-controlled water bath to maintain 

the target temperature (20±1°C) throughout the test period. First-generation daphnids were 

observed daily during the test for mortality, the onset of reproduction, and clinical signs of 

toxicity. Following the onset of reproduction, the numbers of second-generation daphnids were 

counted three times per week (i.e., Monday, Wednesday and Friday). Body lengths and dry 

weights of the surviving first-generation daphnids were measured at the end of the exposure 

period. Observations of the effects of 1-chlorododecane on survival, reproduction and growth 

were used to determine the NOEC, LOEC and MATC. 

Water quality (temperature, dissolved oxygen, pH, hardness, alkalinity and specific conductance) 

was monitored throughout the exposure period. Total organic carbon (TOC) was measured in 

dilution water at test initiation and termination. Water samples also were collected from 

alternating replicate test chambers at the beginning of the test, at weekly intervals during the test 

and at the end of the test to determine concentrations of the test substance. 

Statistical Methods: 

Test endpoints analyzed statistically for first-generation daphnids were survival, reproduction 

(the numbers of live young produced per reproductive day), and growth (length and dry weight). 

Survival data were analyzed using Chi-square and Fisher’s Exact tests. Reproduction and growth 

data were evaluated for normality (Shapiro-Wilk test) and for homogeneity of variance 

(Levene’s test). An analysis of variance (ANOVA) was then used to determine whether or not 

statistically significant differences in reproduction and/or growth existed among the experimental 

groups (p = 0.05); treatments that were significantly different from the control means were 

identified using Dunnett’s t-test (p ≤ 0.05). EC50 values were determined based on cumulative 

reproduction and first-generation mortality/immobility at test termination. The results of the 

statistical analyses were used to aid in the determination of the NOEC, LOEC and maximum 

acceptable toxicant concentration (MATC); however, scientific judgment was used to determine 

if statistical differences were biologically meaningful, and if the data followed a concentrationdependent response. 

Analytical Methods: 

The method used for the analysis of 1-chlorododecane in freshwater samples was based upon 

methodology developed by Wildlife International, Ltd. The analytical method consisted of 

extracting the freshwater samples with ethyl acetate and analyzing by gas chromatography with 

mass selective detection. 

Dilution Water: The water used for culturing and testing was freshwater obtained from a well 

approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water is 

characterized as moderately-hard water. 

Dilution Water Chemistry Parameters: 

Specific Conductance: 355 – 360 μS/cm 

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Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

Hardness: 132 – 140 mg/L as CaCO3

Alkalinity: 176 – 180 mg as CaCO3

pH: 8.2 – 8.2 

Total Organic Carbon: < 1 mg C/L 

RESULTS

Endpoint value(s): 

21-day EC50 (mortality/immobility and reproduction): > 17 μg a.i./L 

21-day NOEC (mortality/immobility and reproduction): 17 μg a.i./L 

21-day NOEC (growth): 7.2 μg a.i./L 

21-day LOEC (mortality/immobility and reproduction): > 17 μg a.i./L 

21-day LOEC (growth): 17 μg a.i./L 

MATC (growth): 11 μg a.i./L

Results remarks: 

Survival and Clinical Observations: 

After 21 days of exposure, survival in the negative and solvent control groups was 95 and 85%, 

respectively. There was no significant difference in survival between the negative and solvent 

control groups. Therefore, the control data were pooled for comparison with the treatment 

groups. Survival in the 1.5, 2.7, 5.7, 7.2 and 17 μg a.i./L treatment groups was 100, 100, 95, 95 

and 95%, respectively, at test termination. There were no statistically significant (p ≤ 0.05) 

reductions in survival at any of the concentrations tested in comparison to the pooled controls 

(Fisher’s Exact test). Consequently, the NOEC for survival was 17 μg a.i./L and the LOEC was 

>17 μg a.i./L. Since less than 50% mortality/immobility was observed among first-generation 

daphnids in any treatment group by test termination, an EC50 value could not be calculated. 

Surviving daphnids in the control groups and in the 1.5, 2.7, 5.7, 7.2 and 17 μg a.i./L treatment 

groups appeared normal at test termination. 

Reproduction: 

With the exception of one replicate in the 7.2 μg a.i./L treatment group and one replicate in the 

17 μg a.i./L treatment group, the first day of brood production in the negative and solvent control 

replicates and in all 1-chlorododecane treatment replicates was Day 7 or 8 of the test, indicating 

that there was no apparent delay in the onset of production at any 1-chlorododecane 

concentration tested. The first day of brood production for Replicate C of the 7.2 μg a.i./L 

treatment group and Replicate A of the 17 μg a.i./L treatment group was Day 11. With the 

exception of two immobile neonates in the 2.7 μg a.i./L treatment at test termination, no 

immobile neonates were noted in the control or treatment groups during the test. No aborted or 

shed eggs were observed during the test. No males or ephippia were produced during the test. 

Adult daphnids in the negative and solvent control groups produced an average of 8.2 and 9.1 

live young per reproductive day, respectively. There was no significant difference in 

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Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

reproduction between the negative and solvent control groups. Therefore, the control data were 

pooled for comparison with the treatment groups. Daphnids in the 1.5, 2.7, 5.7, 7.2 and 17 μg 

a.i./L treatment groups produced an average of 9.4, 9.2, 9.1, 9.4 and 7.7 live young per 

reproductive day, respectively. 

When compared to the pooled control group, Dunnett’s test indicated that there were no 

statistically significant (p > 0.05) differences in reproduction at any of the concentrations tested. 

Consequently, the NOEC for reproduction was 17 μg a.i./L, the highest concentration tested, and 

the LOEC was >17 μg a.i./L. The 21-day EC50 value for reproduction was >17 μg a.i./L. 

Growth: 

Daphnids in the negative control group averaged 5.6 mm in length and 1.08 mg in dry weight, 

while daphnids in the solvent control group averaged 5.5 mm in length and 0.99 mg in dry 

weight. There were no significant differences in the growth parameters between the negative and 

solvent control groups. Therefore, the control data were pooled for comparison with the 

treatment groups. Daphnids in the 1.5, 2.7, 5.7, 7.2 and 17 μg a.i./L treatment groups had mean 

lengths of 5.5, 5.5, 5.6, 5.7 and 5.2 mm, respectively, and mean dry weights of 1.07, 0.95, 1.08, 

1.01 and 0.94 mg, respectively. When compared to the pooled control group, Dunnett’s test 

indicated there was a statistically significant decrease in mean length (p ≤ 0.01) and dry weight 

(p ≤ 0.05) in the 17 μg a.i./L treatment group, and in dry weight (p ≤ 0.05) in the 2.7 μg a.i./L 

treatment group. The decrease in dry weight in the 2.7 μg a.i./L treatment group was slight, not 

associated with a decrease in mean length and was not dose-responsive. Therefore the decrease 

in dry weight for the 2.7 μg a.i./L treatment group was not considered to be treatment-related. 

The decrease in mean length in the 17 μg a.i./L was both strongly significant (p ≤ 0.01) and 

associated with a significant decrease in dry weight. Therefore, impacts on growth at the 17 μg 

a.i./L test concentration were considered to be treatment-related. Consequently, the NOEC for 

growth (based on length and dry weight) was 7.2 μg a.i./L, and the LOEC was 17 μg a.i./L. 

Water Chemistry Measurements: 

Water temperatures measured weekly in the test chambers ranged from 20.0 to 20.3°C. 

Temperatures measured continuously in one negative control chamber were within the 20 ± 1°C 

range specified in the protocol. Dissolved oxygen concentrations in the test chambers remained 

≥68% of saturation (6.1 mg/L) throughout the test period. Measurements of water pH in the test 

chambers ranged from 8.1 to 8.3 throughout the test period. Weekly measurements of specific 

conductance, hardness and alkalinity in the negative control water and in the highest test 

concentration (17 μg a.i./L) were consistent over the test period. TOC in the dilution water at test 

initiation and termination was <1 mg C/L. 

Control Response: 

No statistically significant differences between the negative control and solvent control groups 

were found for any parameter tested (p>0.05); therefore, control data were pooled for 

comparison with the treatment groups. 

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Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

Statistical Results: 

The most sensitive end-point was growth. Compared to the pooled control group, there was a 

statistically significant decrease in mean length (p≤0.05) and dry weight (p≤0.01) in the 17 μg 

a.i./L treatment group, and in dry weight (p≤0.05) in the 2.7 μg a.i./L treatment group. 

The decrease in dry weight in the 2.7 μg a.i./L treatment group was slight, not associated with a 

decrease in mean length and was not dose-responsive, whereas the decrease in mean length in the 

17 μg a.i./L was both strongly significant (p≤0.01) and associated with a significant decrease in 

dry weight. Therefore, unlike impacts on growth at the 17 μg a.i./L test concentration, the 

decrease in dry weight for the 2.7 μg a.i./L treatment group was not considered to be treatmentrelated. Consequently, the NOEC for growth (based on length and dry weight) was 7.2 μg a.i./L, 

and the LOEC was 17 μg a.i./L. 

CONCLUSIONS 

The acute toxicity of the test substance to D. magna has been adequately characterized (Lonza 

Inc.). 

D. magna exposed to 1-chlorododecane at mean measured concentrations of 1.5 to 17 μg a.i./L 

for 21 days showed no significant treatment-related reductions in survival or reproduction at 

concentrations of 17 μg a.i./L and below, or in growth at concentrations of 7.2 μg a.i./L and 

below. Treatment-related reductions in growth (length and dry weight) were apparent at the 17 

μg a.i./L test concentration, the highest concentration tested. Consequently, the NOEC for this 

study, based on the most sensitive endpoint tested (growth) was 7.2 μg a.i./L and the LOEC was 

17 μg a.i./L. The MATC was calculated to be 11 μg a.i./L. The 21-day EC50 value for adult 

mortality/immobility and reproduction was > 17 μg a.i./L. 

STUDY RELIABILITY 

[1] – Reliable without restrictions 

REFERENCE

EPA-HQ-OPPT-2005-0033-0314.7; Gallagher, S.P., Kendall, T.Z., and Krueger, H.O. 2008. A 

Flow-Through Life-Cycle Toxicity Study of 1-Chlorododecane with the Cladoceran (Daphnia 

magna). Wildlife International Ltd. for Lonza Inc. Study No. 289A-166 (unpublished report). 

6 
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: A Flow-Through Life-Cycle Toxicity Study in 1-Chlorododecane with the Cladoceran (Daphnia 

magna)

Test Sponsor: Lonza, Inc. 

Testing Lab: Wildlife International, Ltd. 

Test Standard: ASTM E 1193 

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).