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 Wo rk Assignment Manager

March 5, 2010HPV1 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 22202Draft HPV1 Test Rule – Data Adequacy – T oxicity to Plants (Algae) 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 – T oxicity to Plants (Algae) CAS No. 74-95-3

TOXICITY TO PLANTS (ALGAE)

Title: Dibromomethane: Algal Gr owth Inhibition Test

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 (1984) No. 201, "Alga, Growth Inhibition Test" referenced as Method C.3 of Commi ssion Directive 92/69/EEC (which constitutes Annex V of Council Dir ective 67/548/EEC), US CFR Title 40 Part 797 Section 1050, US EPA Pesticide Assessment Guidel ines, Sub-Division J, Section 122-2, the US EPA Draft Ecological Effects Test Gu ideline OPPTS 850.5400 and ASTM E 1218-04.

Test type : Algal Growth Inhi bition 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 Alg ae 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 solu tion 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 sh aking at a temperature of 24 ± 1°C.

2Draft HPV1 Test Rule – Data Adequacy – T oxicity to Plants (Algae) CAS No. 74-95-3

The control group was maintained under identical cond itions 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 10 6 cells per mL. This suspension was diluted to a cell density of 1.98 x 10 6
cells per mL prior to use. Inoc ulation of 900 mL of test medi um with 4.5 mL of this algal suspension gave an initi al cell density of 10 4 cells per mL and had no significant dilution effect

on the final test concentration.

The flasks were sealed with ground glass stoppe rs and incubated at 24 ± 1°C under continuous illumination (intensity approximately 7000 lux) a nd 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. Aliquot s (0.5 mL) were removed for each replicate culture and the replicates pooled for each concentrati on. Fresh sterile culture medium (100 mL) was

added to ensure that the test concentrations we re 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 vari ance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison pro cedure 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 statisti cally 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 temp erature of 21 ± 1°C under continuous illumination (intensity approxi mately 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 cu lture with the addition of 500 mg/L of sodium

bicarbonate to counteract the incr ease 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 EC 50 (growth): 150 mg/L*; NOEC = 23 mg/L

96-h EC 50 (yield): 95 mg/L; NOEC = 23 mg/L

96-h EC 50 (biomass): 87 mg/L; NOEC= 23 mg/L

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

* It was not possible to calculat e 95% confidence limits for the EC 50 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 grow th rate, yield and biomass integr al data was carried out for the

control and all test concentrations using one way analysis of varian ce incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several

treatments with a control. There were no statis tically significant differen ces between the control and 32 mg/L test concentration (P ≥ 0.05); however all other te st concentrations were significantly different (P < 0.05) and, therefore the NOEC base d on growth rate, yield and biomass integral was 32 mg/L.

Validation Criteria: The following data show that the cell concentrati on of the control culture s 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 cultur es 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 th e control, 10, 32 and 100 mg/L test cultures. However, only a few intact cells were observe d to be present in th e 320 and 1000 mg/L test

cultures.

Observations on test material solubility:

At the start of the test all cont rol and test cultures were observed to be clear colorless solutions. After the 96-hour test period a ll control, 10, 32 and 100 mg/L test cultures were observed to be bright green dispersions while the 320 and 1000 mg/L test culture s were observed to be clear

colorless solutions.

Physico-chemical measurements: The pH values of the control cu ltures were observed to increase fr om pH 7.8 at 0 hours to pH 9.8

- 9.9 at 96 hours. This increas e was considered to be due to the amount of carbon dioxide required by the large number of alga l cells in the log phase of grow th exceeding the transfer rate

of CO 2 from the gaseous phase to the aqueous phase in a sealed test system. In this situation, CO 2 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

4Draft HPV1 Test Rule – Data Adequacy – T oxicity 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 st udy 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 show ed 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 sa mples for unopened vessel analysis at 96 hours. Analysis of these preparations s howed 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 losse s due to volatility. While all efforts were made to prevent losses due to volatilit y, it was not possible to completely close the test system due to the need to open the test vesse ls 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 concentr ations should be used for calculating EC 50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" an alysis 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 EC 50 and NOEC values had no significant effect on th e outcome of the study given that the EC values were of a similar magnitude to those obtai ned based on nominal test concentrations.

Re-Growth Experiment: Re-growth occurred in the cont rol, 10, 32 and 100 mg/L test cu ltures 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 ch loride were within the normal range for this

reference material.

CONCLUSIONS

The effect of the test ma terial on the growth of Pseudokirchneriella subcapitata has been investigated and based on nominal concen trations over a 72-hour period gave an E

r C 50 value of 190 mg/L, an E y C 50 value of 100 mg/L (95% confiden ce limits 84 - 130 mg/L), and an E b C 50 value of 96 mg/L (95% confidence limits 79 - 120 mg/L). Over a 96-hour period, the effect of

5Draft HPV1 Test Rule – Data Adequacy – T oxicity to Plants (Algae) CAS No. 74-95-3

the test material gave an E

r C 50 value of 210 mg/L, an E y C 50 value of 130 mg/L (95% confidence limits 120 - 140 mg/L) and an E b C 50 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 geom etric mean measured test concentrations over a 72-hour period gave an E

r C 50 value of 140 mg/L, an E y C 50 value of 76 mg/L (95% confidence limits 62 - 93 mg/L) and an E b C 50 value of 72 mg/L (95% confidence limits 59 - 87 mg/L). Over a 96-hour period the effect of th e test material gave an E

r C 50 value of 150 mg/L, an E y C 50 value of 95 mg/L (95% confidence limits 87 - 100 mg/L) and an E b C 50 value of 87 mg/L (95% confidence limits 74 - 100 mg/L). The No Observ ed Effect Concentrati on (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 ma intain the stock culture with the addition of 500 mg/L of sodium bicarbonate to coun teract 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 te st 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 ne ed to open the test vess els after each 24-hour period in order to take samples for cell density de termination; the geometric mean measured test

concentrations were used to calculate the EC 50 and NOEC values; it was not possible to calculate

95% confidence limits for the 96-hour ErC 50 value as the data genera ted 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 Hea lth, Safety and Environment.

6Review 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 gr oss, 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) inspecti ons, their dates, and findings were missing or not signed. There is evidence that QA pr ocedures 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 di fferent 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. [ ] 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 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): 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 alga l growth in an enclosed system; analysis of the test preparations at 0 hours showed measured test concentrati ons 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 NO EC 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/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).