SRC TR-10-065 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

Dibromomethane, CAS No. 74-95-3 

Acute Toxicity to Fish 

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 Fish 

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 Fish 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 Fish CAS No. 74-95-3

ACUTE TOXICITY TO FISH 

Title: Dibromomethane: Acute Toxicity to Rainbow Trout (Oncorhynchus mykiss) 

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: OECD Guidelines for Testing of Chemicals (1992) No. 203, “Fish, 

Acute Toxicity Test” referenced as Method C.1 of Commission Directive 92/69/EEC (which 

constitutes Annex V of Council Directive 67/548/EEC). US CFR Title 40 Part 797 Section 

1400; US EPA Pesticide Assessment Guideline, Sub-Division E Section 72-1 and US EPA Draft 

Ecological Effects Test Guideline OPPTS 850.1075 and ASTM E 729-96 Standard Guide for 

Conducting Acute Toxicity Tests on Materials with Fishes, Macroinvertebrates and Amphibians 

Test type: Acute Toxicity to Fish – Semi-Static 

GLP compliant?: Yes 

Year study was performed: 2006 

Test species: Rainbow Trout (Oncorhynchus mykiss). Juvenile rainbow trout used in the study 

were obtained from Brow Well Fisheries Limited, Hebdon, Yorkshire, UK. The fish had a mean 

standard length of 4.9 cm (SD = 0.2) and a mean weight of 1.66 g (SD = 0.30) at the end of the 

definitive test. 

Analytical monitoring: Yes 

Exposure duration: 96 hours

Concentrations tested: 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. 

Test vessels were sealed with minimal headspace due to the suspected volatile nature of the test 

material. A semi-static test regime with daily monitoring of the oxygen concentration was used 

for the definitive test to ensure low oxygen concentrations did not affect the results. 

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

Statistical methods used: The LC50 values were calculated by the trimmed Spearman-Karber 

method. 

Culture conditions: dechlorinated laboratory tap water, single pass water renewal system 

Water temperature: 3.2 to 13.8 °C 

Duration of housing: approximately 15 days 

Mortality during the last 2 weeks of housing: 0% 

Mortality during the adaption period: 0% 

Medical treatment: None 

Diet: commercial trout pellets 

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

filter and partly softened giving water with a total hardness of approximately 150 mg CaCO3/L. 

Renewal interval: 24 hours 

Test water ready for use: 

Total hardness: 124 mg CaCO3/L 

Alkalinity: 94 mg CaCO3/L 

Conductivity: 367 μS/cm 

Total Organic Carbon: < 1 mg/L 

 pH: 7.8 

Particulate matter concentration: 0 .10 mg/L 

Chemical oxygen demand: 2.5 

Volume of water: 25 L 

Loading rate: 0.67 g-bw/L 

Test vessels: completely filled and sealed round glass bottomed glass vessels 

Test temperature: 13.2 to 14.9°C 

Adaption to test water and test temperature: Not indicated 

Photoperiod: 16:8 hours day-night regime with 20-min dawn and dusk transition periods 

Aeration: None 

pH during the test: 7.2 – 7.8 

Dissolved oxygen content: ≤ 10.1 mg O2/L 

Withdrawal of food: 48 hours prior to exposure 

Nominal test concentrations: 10, 18, 32, 56, and 100 mg/L

Observation time: 3, 6, 24, 48, 72 and 96 hours 

Endpoints investigated: mortality and sub-lethal effects 

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

RESULTS

Endpoint value(s): 96-hour LC50 = 45 mg/L; NOEC = 32 mg/L 

Results remarks: The 96-hour LC50 is based on nominal test concentrations. 

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

Range-finding test: The results showed no mortalities at the test concentrations of 1.0 mg/L. 

However, mortalities were observed in the 10 and 100 mg/L test concentrations. After 96 hours 

exposure, two mortalities were observed in the control and a single mortality was observed at the 

10 mg/L test concentration. These mortalities were considered to be due to natural causes and 

possible low oxygen levels due to the vessels and static test conditions used, rather than a true 

toxic effect given that no sub-lethal effects of exposure were observed in the remaining fish and 

no mortalities were observed at the 1.0 mg/L test concentration. 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 highest test concentration resulting in 0% mortality 

to be 32 mg/1, the lowest test concentration resulting in 100% mortality to be 100 mg/L and the 

No Observed Effect Concentration (NOEC) to be 32 mg/L. The No Observed Effect 

Concentration is based upon zero mortalities and the absence of any sub-lethal effects of 

exposure at this concentration. Sub-lethal effects of exposure were observed at the test 

concentration of 56 mg/L. This response was swimming at the surface. The test preparations 

were observed to be clear, colorless solutions throughout the duration of the test. 

Temperatures during the definitive test were observed to be in excess of the range given in the 

protocol of 12 ± 1°C. This deviation was considered not to have affected the outcome or the 

validity of the test as the control fish during testing showed no sub-lethal effects or mortality 

over the test period. 

The oxygen concentration in some of the test vessels for the fresh media was observed to have an 

air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of 

microscopic air bubbles in the media super-saturating the diluent and was considered not to have 

had an impact on the outcome or integrity of the test as no adverse effects were observed. 

Verification of test concentrations: Analysis of the fresh media at 0, 24, 48 and 72 hours showed 

measured test concentrations to range from 82% to 118% of nominal with the exception of the 

10 mg/L test concentration replicates (R1 and R2) at 0 hours which showed measured test 

concentrations of 123% and 124% respectively. As the 10 mg/L test concentration was below the 

No Observed Effect Concentration (NOEC) and the mean measured concentrations over the 96 

hour test period of the two replicates were 114% and 111% of nominal respectively, these results 

were considered not to affect the outcome or validity of the test. In addition, the 32 mg/L test 

concentration replicate (R1) at 72 hours showed a measured concentration of 62% of nominal; 

however, the mean measured concentration was 80% of nominal, therefore this result was 

considered not to affect the outcome or validity of the test. As the majority of the measured 

concentrations were within the 80% to 120% acceptance limits, it was therefore considered 

unnecessary to analyze the frozen duplicate samples. 

Analysis of the old media at 24, 48, 72 and 96 hours showed measured test concentrations to 

range from 80% to 117% of nominal with the exception of the 10 mg/L replicate (R2) at 48 hours 

and the 32 mg/L replicate (R2 ) at 72 hours which showed measured test concentrations of 126% 

and 128% of nominal, respectively. As the mean measured concentrations were 119% and 106% 

of nominal respectively, these results were considered not to affect the outcome or validity of the 

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

test, thus it was considered justifiable to calculate the LC50 values in terms of the nominal test 

concentrations only. 

CONCLUSIONS

The acute toxicity of the test material to the freshwater fish rainbow trout (Oncorhynchus mykiss) 

has been investigated and gave a 96-hour LC50 value of 45 mg/L with 95% confidence limits of 

42 – 48 mg/L. The No Observed Effect Concentration was 32 mg/L. 

STUDY RELIABILITY

 [1] Reliable without restrictions. 

REFERENCE

EPA-HQ-OPPT-2005-0033-0259.1, Durward et al. (2007) Dibromomethane: Acute Toxicity to 

Rainbow Trout (Oncorhynchus mykiss) SPL Project Number 0466/0263, 1-47 pgs. Sponsor: 

Bromine Compounds Ltd. and Albemarle Corporation Health, Safety and Environment. 

5 
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 Rainbow Trout (Oncorhynchus mykiss) 

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).