SRC TR-10-067 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

Dibromomethane, CAS No. 74-95-3 

Acute 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 FG007.0.009.004 

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 Work Assignment Manager 

March 5, 2010 
HPV1 Test Rule – Data Adequacy Assessment 

Dibromomethane, CAS No. 74-95-3 

Acute 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 – Acute Toxicity to Daphnia CAS No. 74-95-3

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 – Acute Toxicity to Daphnia CAS No. 74-95-3

ACUTE TOXICITY TO DAPHNIA 

Title: Dibromomethane: Acute Toxicity to Daphnia magna 

This study is: Adequate Not Adequate 

TEST SUBSTANCE 

 

Identity: Dibromomethane, CAS No. 74-95-3 

Remarks: Clear colorless liquid; Purity: 99.4%; individual samples of dibromomethane were 

supplied by both co-sponsors. These two test materials were then mixed in equal proportions 

and the resulting mixture analyzed to determine its purity. 

METHOD

Method/guideline followed: US CFR Title 40 Part 797 Section 1300: US EPA Pesticide 

Assessment Guideline, Sub-Division E Section 72-2, US EPA Draft Ecological Effects Test 

Guideline OPPTS 850.1010, ASTM E 729-96 and the OECD Guidelines for Testing of 

Chemicals (April 2004) No 202, “Daphnia sp, Acute Immobilization Test” referenced as Method 

C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 

67/548/EEC).

Test type: Acute toxicity to Daphnia - Static

GLP compliant?: Yes

Year study was performed: 2006

Test species: Daphnia magna; derived from in-house laboratory cultures; 1st instar 

Analytical monitoring: Yes 

Exposure duration: 48 hours

Concentrations tested: 

Preliminary range-finding test: 0 (control), 1.0, 10 and 100 mg/L (nominal) 

Definitive test: 0 (control), 10, 18, 32, 56 and 100 mg/L (nominal) 

Test method/conditions remarks: The test concentrations to be used in the definitive test were 

determined by a preliminary range-finding test. Based on the results of the range-finding test, 

the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 

mg/L. A range of five test concentrations was used as it was considered that both a 48-hour EC50

and a No Observed Effect Concentration (NOEC) could be achieved. At the start of the study, 

10 daphnids were placed in each test and control vessel at random, in the test preparations. 

Duplicate test vessels were used for each test and control group. 

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Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Daphnia CAS No. 74-95-3

The test was conducted using glass-stoppered conical flasks with minimal headspace due to the 

volatile nature of the test material. Due to the volatile nature of the test material, it was 

considered that the use of dynamic, continuous flow test conditions was unsuitable as significant 

losses due to volatility may occur. 

Statistical Analysis: The EC50 values and associated confidence limits at 48 hours and the slope 

of the response curve and its standard error were calculated by the maximum-likelihood probit 

method. Probit analysis is used where two or more partial responses to exposure are shown. 

Culture conditions: dechlorinated laboratory tap water 

Water temperature: 18.5 – 18.8 oC 

Diet: algae (Chlorella sp.) 

Test water: dechlorinated laboratory tap water prepared by passage through an activated carbon 

filter and partly softened, giving water with a total hardness of 140 mg CaCO3/L. 

Test water ready for use: 

Particulate matter concentration: 0.50 mg/L 

Total water hardness: 114 mg CaCO3/L 

Alkalinity: 87 mg CaCO3/L 

Conductivity: 428 μS/cm 

Total organic carbon content: 2.5 mg/L 

pH: 7.7 

Test chambers: 300 mL glass-stoppered conical flasks 

Photoperiod: 16:8 hours light-dark regime with 20-minute dawn and dusk transition periods 

Volume of water: 300 mL 

Aeration: None 

Test temperature: 19.6 – 20.4 oC 

pH during the test: 7.7-7.9 

Dissolved O2 during test: 8.4-8.9 mg/L 

Loading rate: ~33 daphnids/L 

Observation time: 24 and 48 hours 

Endpoints investigated: immobilization 

Preparation of the test item: the test material was dissolved directly in dechlorinated tap water. 

RESULTS

Endpoint value(s): The 48-hour EC50 = 66 mg/L; the NOEC was 32 mg/L. 

Results remarks: The 48-hour EC50 value is based on nominal test concentrations. The slope 

and standard error of the response curve at 48 hours was 9.4 (SE = 2.1). Due to the unsuitable 

nature of the data it was not possible to calculate the slope and error of response curve at 24 

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Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Daphnia CAS No. 74-95-3

hours. Test concentrations in excess of 100 mg/L were not used for the test as this is not a 

requirement of the test guidelines. It was considered that the absence of an EC50 value at 24 

hours would not affect the integrity of the study as it is the 48-hour EC50 value which is used for 

classification and labelling purposes. 

Range-finding Test: No immobilization was observed at the test concentrations of 1.0 and 10 

mg/L after 24 and 48 hours. However, immobilization was observed at 100 mg/L. Based on this 

information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive 

test. 

The results of the definitive test showed the lowest test concentration resulting in 100% mortality 

to be greater than 100 mg/L. The No Observed Effect Concentration after 24 and 48 hours 

exposure was 32 mg/L. The No Observed Effect Concentration is based upon zero 

immobilization at this concentration. 

The test media preparations were observed to be clear colorless solutions throughout the duration 

of the test. Analysis of the test preparations at 0 and 48 hours showed measured test 

concentrations to range from 83 to 115% of nominal values and it was considered justifiable to 

calculate the EC50 values in terms of the nominal test concentrations only. 

CONCLUSIONS

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been 

investigated and gave a 48-hour EC50 value of 66 mg/L with 95% confidence limits of 58 – 76 

mg/L. The No Observed Effect Concentration at 48 hours was 32 mg/L. 

STUDY RELIABILITY

[1] Reliable without restrictions 

REFERENCE

EPA-HQ-OPPT-2005-0033-0259.2, Durward et al. (2007) Dibromomethane: Acute Toxicity to 

Daphnia magna, SPL Project Number 0466/0264, 1-34 pgs. Sponsor: Bromine Compounds Ltd. 

and Albemarle Corporation Health, Safety and Environment. 

4 
Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman 

Chemical Name: Dibromomethane 

CAS Registry Number: 74-95-3 

Report Title: Acute Toxicity to Daphnia magna 

Test Sponsor: Bromine Compounds Ltd. and Albemarle Corporation 

Testing Lab: Safepharm Laboratories Limited 

 

Test Standard: ASTM E 729 

Date Study Received by EPA: July 12, 2007 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).