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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia 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 – 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 Te st; 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 (positiv e 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 concentra tions, 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 da ys prior to test initiation in order to achieve equilibrium of the test substance. Two repli cate test chambers (25-L aquaria filled with approximately 22 L of test solution) were tested for each treatment and control. Each replicate

2Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

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

20 D. magna in each treatment and control group.

At test initiation, neonate da phnids were impartially assigned to exposure chambers. Test chambers were indiscriminately positioned in a te mperature-controlled water bath to maintain the target temperature (20±1° C) throughout the test period. Fi rst-generation daphnids were observed daily during the test for mortality, the onset of reproduction, a nd 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 Fr iday). Body lengths and dry weights of the surviving first-generation daphnid s were measured at the end of the exposure period. Observations of the effects of 1-chlo rododecane on survival, reproduction and growth were used to determine the NOEC, LOEC and MATC.

Water quality (temperature, dissolved oxygen, pH, ha rdness, alkalinity and specific conductance) was monitored throughout the exposure period. Total organic carbon (TOC) was measured in

dilution water at test initiation and termina tion. 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 reproductiv e 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-W ilk test) and for homogeneity of variance (Levene’s test). An analysis of variance (ANOVA) was then used to determine whether or not statistically significant differen ces 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). EC 50 values were determined based on cumulative reproduction and first-generation mortality/immobili ty at test termination. The results of the statistical analyses were used to aid in th e determination of the NOEC, LOEC and maximum acceptable toxicant concentration (MATC); however, scientific j udgment was used to determine if statistical differences were biologically mean ingful, and if the data followed a concentrationdependent response.

Analytical Methods: The method used for the analysis of 1-chlor ododecane 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

3Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

Hardness: 132 – 140 mg/L as CaCO 3 Alkalinity: 176 – 180 mg as CaCO 3 pH: 8.2 – 8.2 Total Organic Car bon: < 1 mg C/L

RESULTS

Endpoint value(s): 21-day EC 50 (mortality/immobility and repr oduction): > 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 a nd 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 ne gative and solvent control groups was 95 and 85%, respectively. There was no signifi cant difference in survival be tween 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 μ g a.i./L treatment groups was 100, 100, 95, 95 and 95%, respectively, at test termination. There were no statistic ally significant ( p ≤ 0.05) reductions in survival at any of the concentratio ns tested in comparison to the pooled controls (Fisher’s Exact test). Consequent ly, the NOEC for survival was 17 μ g a.i./L and the LOEC was

>17 μ g a.i./L. Since less than 50% mortality/imm obility was observed among first-generation daphnids in any treatment group by test terminat ion, 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 μ g a.i./L treatment groups appeared normal at test termination.

Reproduction: With the exception of one replicate in the 7.2 μ g a.i./L treatment group an d one replicate in the

17 μ g a.i./L treatment group, the first day of brood pr oduction in the negative and solvent control replicates and in all 1-chlorododecane treatment re plicates was Day 7 or 8 of the test, indicating that there was no apparent delay in the ons et of production at any 1-chlorododecane concentration tested. The first day of brood production for Replicate C of the 7.2 μ g a.i./L treatment group and Replicate A of the 17 μ g a.i./L treatment group was Day 11. With the exception of two immobile neonates in the 2.7 μ g 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 male s 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

4Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

reproduction between the negative an d 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 μ g 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, D unnett’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 μ g a.i./L, the highest concentration tested, and

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

Growth: Daphnids in the negative control group averaged 5.6 mm in lengt h 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 contro l data were pooled for comparison with the treatment groups. Daphnids in the 1.5, 2.7, 5.7, 7.2 and 17 μ g a.i./L treatment groups had mean lengths of 5.5, 5.5, 5.6, 5.7 and 5.2 mm, respect ively, 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 si gnificant decrease in mean length ( p ≤ 0.01) and dry weight ( p ≤ 0.05) in the 17 μ g a.i./L treatment group, and in dry weight ( p ≤ 0.05) in the 2.7 μ g a.i./L treatment group. The decrease in dry weight in the 2.7 μ g a.i./L treatment group was slight, not associated with a decrease in mean length and was not dose-responsive. Therefore th e decrease in dry weight for the 2.7 μ g a.i./L treatment group was not cons idered to be treatment-related. The decrease in mean length in the 17 μ g 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 μ g a.i./L test concentration were considered to be treatment-related. C onsequently, the NOEC for growth (based on length and dry weight) was 7.2 μ g a.i./L, and the LOEC was 17 μ g 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 nega tive control chamber were within the 20 ± 1°C range specified in the protocol. Dissolved oxygen c oncentrations 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 te st period. Weekly measurements of specific conductance, hardness and alkalinity in the nega tive control water and in the highest test concentration (17 μ g 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 diffe rences 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.

5Draft HPV1 Test Rule – Data Adequacy – Chronic Toxicity to Daphnia CAS No. 112-52-7

Statistical Results: The most sensitive end-point was growth. Compar ed 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 μ g a.i./L treatment group, and in dry weight ( p ≤ 0.05) in the 2.7 μ g a.i./L treatment group.

The decrease in dry weight in the 2.7 μ g a.i./L treatment group was s light, not associated with a decrease in mean length and was not dose-responsive, whereas the decrease in mean length in the

17 μ g 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 μ g a.i./L test concentration, the decrease in dry weight for the 2.7 μ g 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 μ g a.i./L,

and the LOEC was 17 μ g 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 a nd below, or in growth at concentrations of 7.2 μg a.i./L and

below. Treatment-related reducti ons in growth (length and dry we ight) were apparent at the 17 μg a.i./L test concentration, th e highest concentrati on 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; Gallaghe r, 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).

6Review 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 St udy 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 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).