SRC TR-15-048

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL):

TOXICITY TO ALGAE

2,4-Hexadienoic acid, (E,E)-, CASRN 110-44-1

Prepared by:

Megan Nelson, William L. Richards, Amy Nguyen, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.4.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Cynthia Bowie, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

August 12, 2015
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 110-

44-1

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TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT .............................................................. 1

B. EVALUATING DATA ADEQUACY ..................................................................................... 1

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 1

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 2

D-1. Toxicity to Aquatic Plants ................................................................................................. 2
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 110-44-1

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a toxicity to algae test for 2,4-hexadienoic acid, (E,E)-, (CASRN 110-

44-1) submitted on May 29, 2015 by Brenntag North America, Inc. to EPA in response to the 

final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of 

Chemicals” (76 FR 1067, January 7, 2011; referred to herein as HPV2; available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0579 as of August 

12, 2015). 

The HPV2 test rule requires C6 aquatic toxicity testing for CASRN 110-44-1. The C6 

requirement consists of toxicity to plants (algae) testing. No other toxicity testing for CASRN 

110-44-1 is required by the HPV2 test rule. 

The May 29, 2015 submission from Brenntag North America, Inc. contained the following study 

report: 

 Softcheck, K.A. 2015. Sorbic acid – 96-hour toxicity test with the freshwater green alga, 

Pseudokirchneriella subcapitata, following OCSPP guideline 850.4500. Smithers 

Viscient, Wareham, MA; Laboratory Project No. 14091.6101. Sponsor: Brenntag North 

America, Inc., Indianapolis, IN. Study report available online at http://www.regulations.

gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0864 as of August 12, 2015.

These newly submitted data are evaluated for adequacy herein. No other toxicity data are 

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

The submitted data are evaluated for adequacy in Section D. The study was summarized 

according to a standard format and assigned a Klimisch reliability code based on its scientific 

merit and conduct, using professional judgment. To determine whether the data were adequate 

to satisfy the C6 testing requirement for CASRN 110-44-1, study details were compared to 

current standard test guidelines to establish whether the study met current testing requirements.1 

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

The study submitted by Brenntag North America, Inc. in the submission dated May 29, 2015 is 

adequate to satisfy the algae toxicity (C6) testing requirement promulgated in the final HPV2 test 

rule for CASRN 110-44-1. 

There are no outstanding HPV2 test rule requirements for toxicity testing of CASRN 110-44-1.

 

1 According to the study report by Softcheck (2015), the toxicity to algae study was conducted following OCSPP 

guideline 850.4500 rather than ASTM guideline E1218-04e1, which is the guideline that is specified on the first page 

of the most-recent study plan that Brenntag North America, Inc. submitted to EPA on March 3, 2015. However, 

EPA considers the use of OCSPP guideline 850.4500 acceptable for conduct of the toxicity to algae test for CASRN 

110-44-1.
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D. DATA FOR SIDS ENDPOINTS

D-1. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata (Softcheck, 2015)

Title: Sorbic acid – 96-hour toxicity test with the freshwater green alga, Pseudokirchneriella 

subcapitata, following OCSPP guideline 850.4500

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: 2,4-Hexadienoic acid, (E,E)-, (CASRN 110-44-1), also known as sorbic acid

Remarks: Lot No. 1401151; Purity: 99.5%; received from GEI Ecology Lab on February 17, 

2015

METHOD

Method/guideline followed: OCSPP 850.4500 (2012) “Algal Toxicity”. 

Test type: Toxicity to plants – algae (static)

GLP compliant?: Yes

Year study was performed: 2015 

Test species: Green algae (Pseudokirchneriella subcapitata); strain: 1648, class Chlorophyceae; 

obtained from the Culture Collection of Algae at The University of Texas (UTEX), Austin, TX 

and maintained at Smithers Viscient

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 

Range-Finding Test: Nominal concentrations: 0 (control), 0.10, 1.0, 10, 100, and 1000 mg/L.

Definitive Test: Nominal concentrations: 0 (control), 6.3, 13, 25, 50, and 100 mg/L; 

corresponding time-weighted average measured concentrations were < LOQ1, 1.7, 3.9, 24, 50,

and 110 mg/L.

 

1 Limit of Quantitation (LOQ) = 0.00747 mg/L
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Test method/conditions remarks: The Algal Assay Protocol (AAP) test medium for the rangefinding and definitive tests was prepared using sterile, deionized water. Analysis of the dilution 

water used for media preparation indicated that there were no pesticides, PCBs, or toxic metals

detected at concentrations “that are considered toxic in any of the water samples analyzed in

agreement with ASTM Guidelines (2007).” [Softcheck 2015, p. 12]

Range-Finding Test Procedure:

A preliminary range-finding test was conducted at nominal concentrations of 0 (control), 0.10, 

1.0, 10, 100, and 1000 mg/L, with two replicates per concentration. The test solutions were 

reported to have been prepared in a manner similar to that described for the definitive test.

Definitive Test Procedure:

Freshwater algae (4 replicates per test concentration, 4 replicates for the control; initial cell 

density of 1 x 104cells/mL) were exposed to nominal concentrations of 0 (control), 6.3, 13, 25, 

50, and 100 mg/L for 96 hours.

Preparation of Test Solutions:

To prepare a stock solution (100 mg/L), an amount of test material (0.2009 g test substance, 

0.1999 g active ingredient) was added to 2000 mL of test medium. This solution was observed 

to be “clear and colorless with a large amount of undissolved material on the bottom of the flask 

and on the solution surface.” [Softcheck 2015, p. 14] Therefore this solution was sonicated for 

10 minutes and then mixed with a stir plate and stir bar for approximately 2.5 hours. “Following 

sonication and mixing, the solution was observed to be clear and colorless with no visible 

undissolved material.” [Softcheck 2015, pp. 14] The stock solution was subsequently diluted to 

produce the desired nominal concentrations. The final solutions were all observed to be clear 

and colorless with no visible undissolved test substance. 

Quality Control Samples:

“Quality control (QC) samples (three at 0-hour and six at 96-hour) were prepared with fresh

AAP medium at nominal concentrations approximating the test concentration range and

remained with the exposure solution samples throughout the analytical process. The three QC 

samples at 0-hour were processed in the same manner as test solution samples. Of the six QC 

samples at 96-hour, three were processed with centrifugation in the same manner as the test 

solution samples and three were processed without centrifugation to evaluate the impact of

centrifugation on test substance recoveries. Analysis of the QC samples was used to judge the

precision and quality control maintained during the analytical process.” [Softcheck 2015, p. 19]

Inoculation of Test Cultures and Environmental Conditions:

Four 250-mL Erlenmeyer flasks were filled with 100 mL of test solution for each treatment level 

and control. The test flasks were covered with stainless steel caps to allow gas exchange. After 

the addition of the test solution, a 1.575-mL inoculum of algal suspension (cell density of 63 x 

104cells/mL) was added to each flask to produce an initial algal cell density of approximately 

1.0 x 104cells/mL. An additional flask that contained test substance at 25 mg/L, but was not 

inoculated with algae, was analyzed at 96 hours to estimate the impact that the presence of algal

biomass had on test substance concentration. 
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“Additional untreated AAP medium was used to prepare the control. Control vessels were 

maintained under the same conditions as the treatment vessels but contained no sorbic acid.” 

[Softcheck 2015, p. 15]

The test vessels were incubated at 24 ± 2°C, under continuous lighting at an intensity range of

approximately 3700 to 4900 lux, and constantly shaken at approximately 100 ± 10 rpm. The 

temperature was continually monitored throughout the study. Water quality parameters 

(conductivity and pH) were measured in the control and test vessels at test initiation. The pH 

was additionally measured at 24, 48, 72, and 96 hours in order to monitor the acidic nature of the 

test substance.

Analytical Monitoring:

Samples were taken from the control and each test group at 0 and 96 hours for quantitative 

analysis. “Samples analyzed at 0-hour were removed from the test and control solutions in the 

volumetric flasks prior to division into the replicate test vessels. Samples analyzed at exposure 

termination (96 hours) were removed from a composite solution from the replicate test vessels of 

each test concentration and the control.” [Softcheck 2015, p. 18] “Prior to processing and 

analysis, all 96-hour exposure solutions were centrifuged to remove algae cells to determine the 

dissolved concentration of test substance...” [Softcheck 2015, p. 18] In addition, “[a]t 96 hours 

of exposure, a sample was removed from the additional replicate flask of the nominal 25 mg/L 

test concentration (replicate F) which did not contain algae and analyzed for sorbic acid 

concentration. The result of the analysis was compared with that obtained for the 96-hour 

analysis of the nominal 25 mg/L solution containing algae to assess the impact that algae had on 

the test substance concentration.” [Softcheck 2015, p. 18] 

Analytical measurements were conducted using a high performance liquid chromatographic 

system equipped with ultraviolet detection (HPLC/UV). A “method validation study was 

conducted prior to the initiation of the definitive test and established an average recovery of 99.5 

± 5.02% from AAP medium.” [Softcheck 2015, p. 19] The mean measured concentrations were 

based on time-weighted average calculations for each treatment level.

Evaluation of Cell Densities and Appearance:

Cell density in each flask was measured at 0, 24, 48, 72, and 96 hours using a hemocytometer 

(Neubauer Improved) and compound microscope. A single sample from each replicate was 

removed to determine cell counts. Microscopic examinations were conducted on one or more 

hemocytometer fields (0.10 x 0.10 cm in surface area, 0.010-cm deep) until at least 400 algae 

cells or four fields were counted. In addition, algal cells were observed at the 24-hour intervals 

for abnormalities. 

Statistical Analysis: 

For determination of the NOEC and LOEC values based on 96-hour yield, average specific 

growth rate, and area under the growth curve, the data were initially checked for normality using 

the Shapiro-Wilk’s Test and for homogeneity of variance using Bartlett’s Test. Based on the 

results of these tests, the data sets for yield and average specific growth rate passed the 

requirement for normality and homogeneity of variance; therefore, Williams’ Multiple 

Comparison Test was used to determine treatment-related effects. Based on the results of the 
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 110-44-1

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Shapiro-Wilk’s and Bartlett’s tests, the data set for area under the growth curve passed the 

requirement for normality but failed the requirement for homogeneity of variance; thus, 

Jonckheere-Terpstra’s Step-Down Test was used to determine treatment-related effects. “All 

statistical determinations were made at the 95% level of certainty, except in the case of ShapiroWilk’s and Bartlett’s Tests, where the 99% level of certainty was applied.” [Softcheck 2015, p. 

21] All statistical analyses were performed using a CETISTM Version 1.8 software package. 

Acceptability criteria based on OCSPP guideline 850.4500: 

1) “at exposure termination, the mean yield and average specific growth rate coefficient of 

variation (CV) of the control should not exceed 15%, and

2) the cell density in the control should increase by a factor of at least 100 times by 

exposure termination (96-hours).” [Softcheck 2015, p. 26]

RESULTS

Endpoint value(s): Based on time-weighted average concentrations in the definitive test:

Response 

Variable EC50 (mg/L)

95% 

Confidence 

Limits (mg/L)

No Observed 

Effect 

Concentration 

(NOEC) (mg/L)

Lowest 

Observed Effect 

Concentration 

(LOEC) (mg/L)

Chronic 

Value 

(ChV) 

(mg/L)1

Yield 10 6.9-10 3.9 24 9.67

Growth Rate 12 11-13 3.9 24 9.67

Biomass 10 8.5-10 3.9 24 9.67

Results remarks: 

Range-Finding Test Results:

“Following 96 hours of exposure, cells exposed to all treatment levels tested and the control were 

observed to be normal. Cell densities in the 0.10, 1.0, 10, 100 and 1000 mg/L treatment levels 

averaged 208.67, 170.50, 187.50, 1.00 and 1.13 x 104cells/mL, respectively. The control 

averaged 186.33 x 104cells/mL. Based on these results and in consultation with the Study 

Sponsor, nominal sorbic acid concentrations of 6.3, 13, 25, 50 and 100 mg/L and a control were 

selected for the definitive exposure.” [Softcheck 2015, p. 22]

Definitive Test Results:

At 0 hours, measured test substance concentrations were 6.4, 13, 25, 51, and 110 mg/L for the 

6.3, 13, 25, 50, and 100 mg/L test concentrations, respectively. At 96 hours, measured test 

substance concentrations were 0.17, 0.56, 23(24)2, 49, and 100 mg/L for the 6.3, 13, 25, 50, and 

100 mg/L test concentrations, respectively. “Since measured concentrations in the low 

concentrations decreased substantially over time, results are calculated as a time-weighted 

average of the measured concentrations in accordance with OSCPP 850.4500 guidance. Based 

on time-weighted average concentrations, treatment levels ranged from 27% to 110% of nominal 

 

1 The ChV value was calculated by the reviewer as the geometric mean of the NOEC and LOEC values.

2 The concentration in parentheses is the result for the additional sample, without algae present, analyzed at 96 

hours. 
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concentrations and defined the treatment levels tested as 1.7, 3.9, 24, 50 and 110 mg/L.” 

[Softcheck 2015, p. 23]

“The analytical result of the 25 mg/L nominal treatment level with algae present was 23 mg/L at 

96 hours... The equivalent test solution without algae present resulted in a recovery of 24 mg/L 

at 96 hours; however, this treatment had nearly 100% inhibition of algal growth. Consequently 

the impact of algae on the test substance concentration could not be assessed within this 

treatment.” [Softcheck 2015, pp. 23-24]

“Analysis of the uncentrifuged quality control (QC) samples resulted in measured concentrations 

which were consistent with the predetermined recovery range, ranging from 98.0 to 107% (N = 

6) of the nominal fortified levels (3.00, 25.0 and 100 mg/L). Analysis of the centrifuged quality 

control samples resulted in measured concentrations which ranged from 97.8 to 104% (N = 3) of 

the nominal fortified levels (3.00, 25.0 and 100 mg/L). Based on these results, it was established 

that the appropriate precision and quality control was maintained during processing and that 

centrifugation of the test samples did not have a negative impact on their quantitation.” 

[Softcheck 2015, p. 24]

Biological results: “The 96-hour cell density in the control averaged 219.08 x 104cells/mL. Cell 

densities in the 1.7, 3.9, 24, 50 and 110 mg/L treatment levels averaged 219.88, 216.79, 2.81, 

1.38 and 0.81 x 104cells/mL, respectively.” [Softcheck 2015, p. 24] Significant reductions in 

growth rate, yield, and biomass (area under the growth curve) were detected at time-weighted 

average concentrations ≥ 24 mg/L compared to control data. Therefore, the 96-hour NOEC and 

LOEC values were determined to be 3.9 and 24 mg/L, respectively, based on growth rate, yield,

and biomass. For growth rate inhibition, “[b]ased on the time-weighted average concentrations, 

the 96-hour EC50 value for average specific growth rate was determined to be 12 mg/L, with 

95% confidence limits of 11 to 13 mg/L.” [Softcheck 2015, p. 25] For inhibition of yield, 

“[b]ased on the time-weighted average concentrations, the 96-hour EC50 value for yield was 

determined to be 10 mg/L, with 95% confidence limits of 6.9 to 10 mg/L.” [Softcheck 2015, p. 

24] For inhibition of biomass (area under the growth curve or AUGC), “[b]ased on the timeweighted average concentrations, the 96-hour EC50 value for AUGC was determined to be 10 

mg/L, with 95% confidence limits of 8.5 to 10 mg/L.” [Softcheck 2015, p. 25]

Cell densities and mean percentage values for inhibition of growth rate, yield, and biomass 

relative to the control at each time-weighted average concentration were as follows:

Time-Weighted 

Average 

Concentration 

(mg/L)

Mean Cell Densities (x 104

cells per mL) Inhibition Values (%)

0 Hours 96 Hoursa Growth Rate Yield Biomass

Control (0) 1.00 219.08 

(35.25)c

- -

-

1.7 1.00 219.88 (38.63) 0 0 [1]b

3.9 1.00 216.79 (40.00) 0 1 [4]b

24 1.00 2.81 (0.75) 81 99 98

50 1.00 1.38 (0.75) 96 100 100

110 1.00 0.81 (0.66) 108 100 100
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a Cell densities at 96 hours are the means of four replicates per concentration.

b [Increase in biomass compared to controls]

c Standard deviation (SD) in parentheses

Observations of test conditions: “The pH of the control solution was 7.2 at test initiation and 

increased to 9.4 after 96 hours. The pH of the test solutions ranged from 4.2 to 6.7 at test 

initiation. The solution pH was inversely proportional to the test concentration, decreasing with 

increasing test substance concentration. At 96 hours of exposure, the pH of the test solutions 

ranged from 4.3 to 9.3, increasing relative to biomass increase in the 6.3 and 13 mg/L [nominal] 

treatment levels and remaining largely unchanged from a pH of approximately 5 in the three high 

treatment levels (25, 50 and 100 mg/L) where nearly 100% inhibition of algal growth was 

observed. The increase in pH relative to biomass increase during the exposure is common in 

static algal cultures and is due to photosynthesis by the algae. Conductivity of the treatment and 

control solutions ranged from 90 to 120 μS/cm, increasing slightly as the concentration of test 

substance increased. Continuous temperature monitoring established that the temperature ranged 

from 23 to 24 °C throughout the exposure period. The photosynthetically-active radiation (PAR) 

of the test area ranged from 51 to 66 μE/m2/S throughout the exposure period. Light intensity 

(lux) of the test area at test initiation ranged from 4300 to 4900 lux. The shaking rate was 

maintained throughout the exposure at a constant rate of 100 rpm.” [Softcheck 2015, pp. 22-23]

Acceptability Criteria: 

The definitive test met the following criteria for acceptability prescribed by the protocol:

 The 0- to 96-hour average specific growth rate CV for the control was 3.0% (criterion: 

≤ 15%).

 The 96-hour cell density in the control was 219.08 x 104cells/mL (criterion: ≥ 100 x 104

cells/mL).

The definitive test did not meet the following criterion for acceptability prescribed by the 

protocol:

 The 0- to 96-hour average yield CV for the control was 16% (criterion: ≤ 15%).

CONCLUSIONS

Based on time-weighted average concentrations, the 96-hour EC50 values for inhibition of 

growth, yield, and biomass were 12, 10, and 10 mg/L, respectively. The 96-hour NOEC and 

LOEC were 3.9 and 24 mg/L, respectively. Based on this information, the ChV was calculated 

by the reviewer to be 9.67 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. The 0- to 96-hour average yield CV for the control was 16%, 

which exceeded the criterion of ≤ 15%. 

REFERENCE

Softcheck, K.A. 2015. Sorbic acid – 96-hour toxicity test with the freshwater green alga, 

Pseudokirchneriella subcapitata, following OCSPP guideline 850.4500. Smithers Viscient,

Wareham, MA; Laboratory Project No. 14091.6101. Sponsor: Brenntag North America, Inc., 
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN 110-44-1

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Indianapolis, IN. Study report available online at http://www.regulations.gov/#!document

Detail;D=EPA-HQ-OPPT-2007-0531-0864 as of August 12, 2015.

Text in quotes is taken directly from Softcheck (2015).