SRC TR-10-045 

HPV1 TEST RULE STUDY – DATA ADEQUACY ASSESSMENT 

2,4-Hexadienoic acid, CAS No. 110-44-1 

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 

2,4-Hexadienoic acid, CAS No. 110-44-1 

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 – Toxicity to Fish CAS No. 110-44-1

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. 

1 
Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Fish CAS No. 110-44-1

ACUTE TOXICITY TO FISH 

Title: Fathead minnow 96-h acute survival test 

This study is: Adequate Not Adequate 

The study is considered to be inadequate because the test report lacked details concerning test 

substance purity, GLP compliance, weight/length and acclimatization of test organisms, loading 

rate, and analytical monitoring. 

TEST SUBSTANCE 

 

Identity: 2,4-Hexadienoic acid, CAS No. 110-44-1; purity not specified (see Study Reliability 

section for comment) 

Remarks: Sorbic Acid, Lot # 200710051; Water Solubility: slightly soluble (1000 mg/L at 

25 oC); Log Kow: 0.62; Melting Point: 133.9 oC; Boiling Point: 228 oC

METHOD

Method/guideline followed: The study was performed according to ASTM E 729-96 (2002) 

and EPA-821-R-02-012 (2002). 

Test type: Acute Fish Toxicity- Static Renewal 

GLP compliant?: Not Specified 

Year study was performed: 2008 

Test species: Pimephales promelas. Larval (6-day old) fathead minnows used in this study were 

obtained from the Aquatic Bio Systems Inc., 1300 Blue Spruce Drive Suite C, Fort Collins, 

Colorado 80524. 

Analytical monitoring: Not Specified 

Exposure duration: 96-hours

Concentrations tested: 0 (dilution water), 25, 50, 100, 175 and 250 mg/L 

Test method/conditions remarks: 

Statistical methods used: The LC50 value was determined using the Trimmed Spearman-Karber 

method 

Dilution water: Lab water; moderately hard reconstituted water 

Feeding: before 48 hour solution renewal 

Food: Artemia sp. 

Exposure Chamber: 9 oz. disposable plastic cup 

2 
Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Fish CAS No. 110-44-1

Volume of exposure chamber: 250 mL 

Number of animals exposed/chamber: 10 

Number of replicates/treatment: 4 

Test Conditions: 

Dissolved O2: 7.0 – 7.3 mg/L 

pH: 4.85 – 8.00 

Conductivity (μmho/cm): 330 – 361 

Test Temperature: 19.5 – 20.5 oC 

Water chemistry (analysis of control and 250 mg/L samples): 

Temperature: 20oC (sorbic acid); 25 oC (reconstituted water) 

Total Hardness: 93 – 102 mg CaCO3/L 

 pH: 4.80 – 8.08 

Alkalinity: 1.8 – 63 mg CaCO3/L 

Conductivity: 314 – 325 μmho/cm 

Total Dissolved Solids: 154 mg/L (from sorbic acid sample only) 

Dissolved Oxygen: 7.0 – 7.5 mg O2/L 

Ammonia: 0.03 mg NH3/L 

Un-ionized Ammonia: < 0.1 mg NH3/L 

Total Residual Chlorine: 0.03 mg/L 

Control performance criteria: 90% survival required 

Observation time: 24, 48, 72 and 96 hours 

Endpoints investigated: Mortality 

RESULTS

Endpoint value(s): The 96-hour LC50 value for fathead minnows was 117.5 mg/L (95% C.I. 

107.9 – 127.9 mg/L). 

Results remarks: Percent survival at 96 hours was 90%, 87.5%, 87.5%, 72.5%, 0% and 0% at 

nominal concentrations of 0, 25, 50, 100, 175 and 250 mg/L, respectively. Control performance 

criteria were met. Mortalities at the two highest concentrations appear to be mainly related to pH 

(measured on day 1 as 5.35 for 175 mg/L sorbic acid and 4.84 for 250 mg/L sorbic acid). 

CONCLUSIONS

The 96-hour LC50 = 117.5 mg/L (nominal). 

STUDY RELIABILITY

[3] Not reliable − The test report lacked study details including test methods and procedures, test 

substance purity information, GLP compliance, weight/length and acclimatization of test 

organisms, loading rate and whether or not analytical monitoring was conducted. 

3 
Draft HPV1 Test Rule – Data Adequacy – Acute Toxicity to Fish CAS No. 110-44-1

REFERENCE

EPA-HQ-OPPT-2005-0033-0313. Wego Chemical & Mineral Corp (WEGO). Fathead Minnow 

96-h Acute Survival Test. Performing Laboratory: GEI Consultants Inc./Chadwick Ecological 

Division. Report Compiled by Garden State Analytix. Test No. 09-7879-6209. (2008). 

4 
Review of TSCA Section 4 Final Study Report

CITB Project Manager: Catherine Roman 

Chemical Name: 2,4-Hexadienoic acid 

CAS Registry Number: 110-44-1 

Report Title: Fathead minnow 96-h Acute Survival Test 

Test Sponsor: Wego Chemical & Mineral Corp (WEGO) 

Testing Lab: GEI Consultants Inc./Chadwick Ecological Division 

Test Standard: ASTM E 729 

Date Study Received by EPA: August 13, 2008 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. 

 [C] 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. 

 [D] 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. 

 [A] 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. 

 [C] 6. There is evidence that Good Laboratory Practice Standards (GLPS) were inadequately followed. 

 [C] 7. The test, control, or reference substances are different from that specified in the section 4 test rule or 

Enforceable Consent Agreement (ECA). 

 [A] 8. Animal identification is inadequate or unclear; use of unhealthy animals or other factors (e.g., deviations 

from temperature, lighting, dosing) are apparent. 

 [A] 9. The final study report inadequately describes the study method. 

 [A] 10. The final study report inadequately describes the test system. 

 [A] 11. The final study report inadequately describes the dosage information. 
[A] 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 [ ] 

 deficiencies (list in notes below). 

 3. The study conduct was significantly deficient or questionable, (give [X] 

 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. [ ] 

 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). [X] 

Reviewer Comments (continue on separate page, if needed): 

Part I, Question 2: The loading rate (g fish/L) is not specified. Analytical monitoring of the test substance 

concentration is not described and measured concentrations are not provided. Therefore, it cannot be determined 

whether or not the requirements for loading rate and analytical monitoring were violated. 

Part I, Question 5: The Statement of Quality Assurance is not signed. 

Part I, Question 6: GLP compliance is not stated 

Part I, Question 7: Purity of the test substance is not provided. 

Part I, Question 8: Weight/length and acclimatization of test animals are not specified. 

Part I, Questions 9 and 10: The study report does not adequately describe the study method or test system, as 

described in other comments. 

Part I, Question 11: Analytical measurements of the test substance are not provided. 

Part I, Question 12: Water quality data are not provided for each replicate. 

Part II, Study Conduct: The test report does not provide sufficient information, as described above. 

Part II, Data Reliability: Given that the total dissolved solids concentration is 154 mg/L at the treatment 

concentration of 250 mg sorbic acid/L, it appears that the test substance did not dissolve completely in solution. 

Additionally, because insufficient test details have been provided, it is unclear if the test results would be the same if 

the test was repeated. 

Part II, Data Utility: Because the absence of analytical chemistry data prevents the determination of an accurate 

LC50, the data are not useful. 

Reviewer Name/Title/Branch/Division/Office: Justine Von Runnen/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).