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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Fish CAS No. 74-95-3

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 – 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%; indi vidual samples of dibromomethane were supplied by both co-sponsors. These two test ma terials were then mixed in equal proportions and the resulting mixture anal yzed 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, Macroi nvertebrates 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 tr out used in the study

were obtained from Brow Well Fisheries Limite d, 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.

2Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Fish CAS No. 74-95-3

Statistical methods used: The LC 50 values were calculated by the trimmed Spearman-Karber

method. Culture conditions: dechlorinated laboratory ta p 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 la boratory tap water prepared by pa ssage through an activated carbon filter and partly softened gi ving water with a total hardne ss of approximately 150 mg CaCO 3 /L.

Renewal interval: 24 hours

Test water ready for use: Total hardness: 124 mg CaCO 3 /L Alkalinity: 94 mg CaCO 3 /L Conductivity: 367 μ S/cm Total Organic Carbon: < 1 mg/L pH: 7.8

Particulate matter concen tration: 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 O 2 /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 wa s dissolved directly in dechlorinated tap water.

RESULTS

Endpoint value(s): 96-hour LC 50 = 45 mg/L; NOEC = 32 mg/L

Results remarks: The 96-hour LC 50 is based on nominal te st concentrations.

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

Range-finding test: The results showed no mortalities at th e test concentrations of 1.0 mg/L. However, mortalities were observed in the 10 an d 100 mg/L test concentrations. After 96 hours exposure, two mortalities were observed in the co ntrol and a single mortality was observed at the 10 mg/L test concentration. These mortalities we re considered to be due to natural causes and possible low oxygen levels due to the vessels and st atic test conditions used, rather than a true toxic effect given that no sub-le thal effects of exposure were obs erved in the remaining fish and

no mortalities were observed at th e 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 high est test concentration resulting in 0% mortality to be 32 mg/1, the lowest test concentration re sulting 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 e ffects of exposure were observed at the test concentration of 56 mg/L. This response was swim ming at the surface. The test preparations were observed to be clear, colorless so lutions 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 consid ered not to have affected the outcome or the validity of the test as the control fish during te sting showed no sub-lethal effects or mortality over the test period.

The oxygen concentration in some of the test vesse ls 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-saturati ng 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 no minal with the exception of the 10 mg/L test concentr ation replicates (R 1 and R 2 ) 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 re spectively, these results

were considered not to affect the outcome or va lidity of the test. In addition, the 32 mg/L test concentration replicate (R 1 ) at 72 hours showed a measured con centration of 62% of nominal; however, the mean measured concentration wa s 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 hour s showed measured test concentrations to range from 80% to 117% of nominal with the exception of the 10 mg/L replicate (R 2 ) at 48 hours and the 32 mg/L replicate (R 2 ) at 72 hours which showed measured test concentrations of 126% and 128% of nominal, respectively. As the mean measured concentratio ns were 119% and 106% of nominal respectively, these results were consider ed not to affect the out come or validity of the

4Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Fish CAS No. 74-95-3

test, thus it was considered justifiable to calculate the LC 50 values in terms of the nominal test concentrations only.

CONCLUSIONS

The acute toxicity of the te st material to the freshw ater fish rainbow trout ( Oncorhynchus mykiss ) has been investigated and gave a 96-hour LC 50 value of 45 mg/L with 95% confidence limits of 42 – 48 mg/L. The No Observed E ffect Concentration was 32 mg/L.

STUDY RELIABILITY

[1] Reliable wit hout 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 Corpor ation Health, Safety and Environment.

5Review 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 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).