SRC TR-10-068 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

Dibromomethane, CAS No. 74-95-3 

Toxicity to Plants (Algae) 

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 

Toxicity to Plants (Algae) 

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 – Toxicity to Plants (Algae) 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 – Toxicity to Plants (Algae) CAS No. 74-95-3

TOXICITY TO PLANTS (ALGAE) 

Title: Dibromomethane: Algal Growth Inhibition Test 

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 (1984) No. 201, 

"Alga, Growth Inhibition Test" referenced as Method C.3 of Commission Directive 92/69/EEC 

(which constitutes Annex V of Council Directive 67/548/EEC), US CFR Title 40 Part 797 

Section 1050, US EPA Pesticide Assessment Guidelines, Sub-Division J, Section 122-2, the US 

EPA Draft Ecological Effects Test Guideline OPPTS 850.5400 and ASTM E 1218-04. 

Test type: Algal Growth Inhibition Test- Static

GLP compliant?: Yes 

Year study was performed: 2007 

Test species: Green Alga, Pseudokirchneriella subcapitata (strain CCAP 278/4); liquid cultures 

obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine 

Laboratory, Oban, Argyll, Scotland.

Analytical monitoring: Yes 

Exposure duration: 96-hours

Concentrations tested: 

Range-finding test: 0 .10, 1 .0, 10, 100 and 1000 mg/L 

Definitive test: 10, 32, 100, 320 and 1000 mg/L (mean measured concentrations of 11, 23, 77, 

230 and 830 mg/L, respectively) 

Test method/conditions remarks: Following a preliminary range-finding test, 

Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material at 

concentrations of 10, 32, 100, 320 and 1000 mg/L (three replicate flasks per concentration) for 

96 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. 

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Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 74-95-3

The control group was maintained under identical conditions but not exposed to the test material. 

Due to the volatile nature of the test material, an additional replicate was prepared for the control 

and each treatment group at 0 hours and incubated alongside the test to provide samples at 96 

hours from unopened vessels. 

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell 

density of 6 .77 x 106

 cells per mL. This suspension was diluted to a cell density of 1.98 x 106

cells per mL prior to use. Inoculation of 900 mL of test medium with 4.5 mL of this algal 

suspension gave an initial cell density of 104

 cells per mL and had no significant dilution effect 

on the final test concentration. 

The flasks were sealed with ground glass stoppers and incubated at 24 ± 1°C under continuous 

illumination (intensity approximately 7000 lux) and constantly shaken at approximately 150 rpm 

for 96 hours. 

Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined. 

A re-growth experiment was performed after 96 hours exposure to determine the algicidal or 

algistatic effect of the test material. Aliquots (0.5 mL) were removed for each replicate culture 

and the replicates pooled for each concentration. Fresh sterile culture medium (100 mL) was 

added to ensure that the test concentrations were reduced to below the inhibiting level. The subcultures were incubated at 24 ± 1°C for a period of 192 hours. 

Statistical Analysis: One way analysis of variance incorporating Bartlett's test for homogeneity 

of variance and Dunnett's multiple comparison procedure for comparing several treatments with 

a control was carried out on the area under the growth curve data at 96 hours for the control and 

all test concentrations to determine any statistically significant differences between the test and 

control groups. 

Culture conditions: Cultures were maintained in the laboratory by the periodic replenishment of 

culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1°C under 

continuous illumination (intensity approximately 7000 lux) and constant aeration. 

Culture medium: The culture medium used for both the range-finding and definitive tests was the 

same as that used to maintain the stock culture with the addition of 500 mg/L of sodium 

bicarbonate to counteract the increase in pH due to algal growth in an enclosed system. 

Preparation of test substance: The test material was dissolved directly in culture medium. 

RESULTS

Endpoint value(s): The following results were determined from the data based on the geometric 

mean measured test concentrations:

96-h EC50 (growth): 150 mg/L*; NOEC = 23 mg/L 

96-h EC50 (yield): 95 mg/L; NOEC = 23 mg/L 

96-h EC50 (biomass): 87 mg/L; NOEC= 23 mg/L 

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Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 74-95-3

* It was not possible to calculate 95% confidence limits for the EC50 values as the data generated 

did not fit the models available for the calculation of confidence limits. 

Results remarks: 

Range-Finding Test: 

The results showed no significant effect on growth at the test concentrations of 0.10, 1.0 and 10 

mg/L. However, growth was observed to be reduced at 100 and 1000 mg/L. Based on this 

information test concentrations of 10, 32, 100, 320, and 1000 mg/L were selected for the 

definitive test. 

Definitive Test: 

Statistical analysis of the growth rate, yield and biomass integral data was carried out for the 

control and all test concentrations using one way analysis of variance incorporating Bartlett's test 

for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several 

treatments with a control. There were no statistically significant differences between the control 

and 32 mg/L test concentration (P ≥ 0.05); however all other test concentrations were 

significantly different (P < 0.05) and, therefore the NOEC based on growth rate, yield and 

biomass integral was 32 mg/L. 

Validation Criteria: 

The following data show that the cell concentration of the control cultures increased by a factor 

of 110 after 72 hours and 205 after 96 hours. The coefficient of variation for average specific 

growth rate for the control cultures over the test period (0 - 96 h) was 4%. The coefficient of 

variation for yield for the control cultures over the test period (0 - 96 h) was 14%. 

Observations on Cultures: 

All test and control cultures were inspected microscopically at 96 hours. After 96 hours there 

were no abnormalities detected in any of the control, 10, 32 and 100 mg/L test cultures. 

However, only a few intact cells were observed to be present in the 320 and 1000 mg/L test 

cultures. 

Observations on test material solubility: 

At the start of the test all control and test cultures were observed to be clear colorless solutions. 

After the 96-hour test period all control, 10, 32 and 100 mg/L test cultures were observed to be 

bright green dispersions while the 320 and 1000 mg/L test cultures were observed to be clear 

colorless solutions. 

Physico-chemical measurements: 

The pH values of the control cultures were observed to increase from pH 7.8 at 0 hours to pH 9.8 

- 9.9 at 96 hours. This increase was considered to be due to the amount of carbon dioxide 

required by the large number of algal cells in the log phase of growth exceeding the transfer rate 

of CO2 from the gaseous phase to the aqueous phase in a sealed test system. In this situation, CO2

required for photosynthesis and growth would be derived from bicarbonate in solution which 

results in an increase in the pH of the culture. The increase in pH after 96 hours was in excess of 

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Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 74-95-3

that recommended in the Test Guidelines (1.5 pH units after 96 hours). This was considered to 

have had no adverse effect on the results of the study given that the increase in cell concentration 

in the control cultures exceeded the validation criterion given in the Test Guidelines. 

Verification of test concentrations: 

Analysis of the test preparations at 0 hours showed measured test concentrations to be near 

nominal with the exception of the 32 mg/L test sample which showed a measured concentration 

of 79% of nominal. Analysis of the test preparations at 96 hours showed a slight decline in 

measured test concentrations in the range 64 to 101% of nominal. 

Due to the volatile nature of the test material, additional test replicates were prepared at 0 hours 

and incubated alongside the test to provide samples for unopened vessel analysis at 96 hours. 

Analysis of these preparations showed measured test concentrations to range from 81 to 106% of 

nominal. As these measured concentrations were near nominal it was considered that the decline 

seen in the 96 hour test samples was through losses due to volatility. While all efforts were made 

to prevent losses due to volatility, it was not possible to completely close the test system due to 

the need to open the test vessels after each 24-hour period in order to take samples for cell 

density determination. 

Current regulatory advice is that in cases where a decline in measured concentrations is 

observed, geometric mean measured concentrations should be used for calculating EC50 values. 

It was therefore considered justifiable to base the results on the geometric mean measured test 

concentrations in order to give a "worst case" analysis of the data. The geometric mean measured 

test concentrations were determined to be: 11, 23, 77, 230 and 830 mg/L, expressed at 110%, 

72%, 77%, 72% and 83% of the nominal test concentrations (10, 32, 100, 320 and 1000 mg/L), 

respectively. 

The use of the geometric mean measured test concentrations in the calculation of the EC50 and 

NOEC values had no significant effect on the outcome of the study given that the EC values 

were of a similar magnitude to those obtained based on nominal test concentrations. 

Re-Growth Experiment: 

Re-growth occurred in the control, 10, 32 and 100 mg/L test cultures after 48 hours, in the 320 

mg/L test cultures after 168 hours and in the 1000 mg/L test cultures after 192 hours. These 

results indicated that the test material was algistatic in effect. 

Positive Control: 

The results from the positive control with zinc chloride were within the normal range for this 

reference material. 

CONCLUSIONS

The effect of the test material on the growth of Pseudokirchneriella subcapitata has been 

investigated and based on nominal concentrations over a 72-hour period gave an ErC50 value of 

190 mg/L, an EyC50 value of 100 mg/L (95% confidence limits 84 - 130 mg/L), and an EbC50

value of 96 mg/L (95% confidence limits 79 - 120 mg/L). Over a 96-hour period, the effect of 

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Draft HPV1 Test Rule – Data Adequacy – Toxicity to Plants (Algae) CAS No. 74-95-3

the test material gave an ErC50 value of 210 mg/L, an EyC50 value of 130 mg/L (95% confidence 

limits 120 - 140 mg/L) and an EbC50 value of 110 mg/L (95% confidence limits 96 - 140 mg/L). 

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

The effect of the test material based on the geometric mean measured test concentrations over a 

72-hour period gave an ErC50 value of 140 mg/L, an EyC50 value of 76 mg/L (95% confidence 

limits 62 - 93 mg/L) and an EbC50 value of 72 mg/L (95% confidence limits 59 - 87 mg/L). Over 

a 96-hour period the effect of the test material gave an ErC50 value of 150 mg/L, an EyC50 value 

of 95 mg/L (95% confidence limits 87 - 100 mg/L) and an EbC50 value of 87 mg/L (95% 

confidence limits 74 - 100 mg/L). The No Observed Effect Concentration (NOEC) was 23 mg/L. 

A re-growth experiment showed the test material was algistatic in effect. 

STUDY RELIABILITY

[2] Reliable with restrictions ─ The culture medium used for both the range-finding and 

definitive tests was the same as that used to maintain the stock culture with the addition of 500 

mg/L of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed 

system; the increase in pH after 96 hours was in excess of that recommended in the Test 

Guidelines (1.5 pH units after 96 hours); analysis of the test preparations at 0 hours showed 

measured test concentrations to be near nominal with the exception of the 32 mg/L test sample 

which showed a measured concentration of 79% of nominal; analysis of the test preparations at 

96 hours showed a slight decline in measured test concentrations in the range 64 to 101% of 

nominal; while all efforts were made to prevent losses due to volatility, it was not possible to 

completely close the test system due to the need to open the test vessels after each 24-hour 

period in order to take samples for cell density determination; the geometric mean measured test 

concentrations were used to calculate the EC50 and NOEC values; it was not possible to calculate 

95% confidence limits for the 96-hour ErC50 value as the data generated did not fit the models 

available for the calculation of confidence limits. 

REFERENCE

EPA-HQ-OPPT-2005-0033-0254.3, Mullee et al. (2007) Dibromomethane: Algal Growth 

Inhibition Test, SPL Project Number 0466/0265, 1-68 pgs. Sponsor: Bromine Compounds Ltd 

and Albemarle Corporation Health, Safety and Environment.

6 
Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman 

Chemical Name: Dibromomethane 

CAS Registry Number: 74-95-3 

Report Title: Dibromomethane: Algal Growth Inhibition Test

Test Sponsor: Bromine Compounds Ltd. and Albemarle Corporation 

Testing Lab: Safepharm Laboratories Limited 

 

Test Standard: ASTM E 1218 

 

Date Study Received by EPA: June 07, 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. 

[A] 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. [ ] 

 2. The study was generally well-conducted, with some minor [X] 

 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. [ ] 

 5. Appropriate methods were used to determine [X] 

 statistical significance. 

 6. If repeated, the test results would be expected to be the [ ] 

 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): The culture medium used for both the range-finding 

and definitive tests was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium 

bicarbonate to counteract the increase in pH due to algal growth in an enclosed system; analysis of the test 

preparations at 0 hours showed measured test concentrations to be near nominal with the exception of the 32 mg/L 

test sample which showed a measured concentration of 79% of nominal. Analysis of the test preparations at 96 hours 

showed a slight decline in measured test concentrations in the range 64% to 101 % of nominal. Whilst all efforts 

were made to prevent losses due to volatility it was not possible to completely close the test system due to the need 

to open the test vessels after each 24-hour period in order to take samples for cell density determination; the 

geometric mean measured test concentrations were used to calculate the EC50 and NOEC values; therefore, it was 

not possible to calculate 95% confidence limits for the 96-hour ErC50 value as the data generated did not fit the 

models available for the calculation of confidence limits. 

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).