SRC TR-16-017

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL):

TOXICITY TO ALGAE

D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt, CASRN 6381-77-7

Prepared by:

Amy Nguyen, William Richards, and Teresa Manyin

Defense and Environmental Solutions Division

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-12-003

Task FA552.4.011.001

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

April 18, 2016
Final HPV2 Test Rule Data Adequacy Review (April 2016) CASRN 6381-77-7

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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 ........ 2

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 3

D-1. Toxicity to Aquatic Plants ................................................................................................. 3
Final HPV2 Test Rule Data Adequacy Review (April 2016) CASRN 6381-77-7

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a toxicity to algae study of CASRN 6381-77-7 (D-erythro-hex-2-enonic 

acid, .gamma.-lactone, monosodium salt; sodium erythorbate) submitted on March 5, 2016 by 

Greenwich Chemical Consulting, Inc. (on behalf of Univar USA Inc.) to EPA1in 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 test rule; available online 

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

April 18, 2016). 

The HPV2 test rule requires the following toxicological tests for CASRN 6381-77-7: 

C1 (consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia, and toxicity to 

algae testing] if log Kow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae 

testing] if log Kow ≥ 4.2). Based on an experimentally measured log Kow of < -1.552, which is 

< 4.2, the C1 toxicity testing requirement is considered to include acute toxicity to fish, acute 

toxicity to Daphnia, and toxicity to algae testing. 

The March 5, 2016 submission from Greenwich Chemical Consulting, Inc. contained the 

following study report: 

 Vryenhoef, H. (Study Director). 2016. D-erythro-Hex-2-enonic acid, .gamma.-lactone, 

monosodium salt [CAS RN 6381-77-7]: Algal growth inhibition test. Performing 

Laboratory: Envigo Research Limited, Derbyshire, UK. Study No. 41401175. Sponsor: 

Univar USA Inc.

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 data were adequate to 

satisfy test rule requirements, study details were compared to OECD guidelines.3 

 

1 Study submitted by email from Mr. John V. Massingale (Greenwich Chemical Consulting, Inc.) to Mr. John 

Schaeffer (EPA) on March 5, 2016.

2 Walker, J.A. (Study Director). 2015. D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 

6381-77-7]: Determination of partition coefficient (n-octanol/water). Performing Laboratory: Envigo Research 

Limited, Derbyshire, UK. Sponsor: Univar USA Inc. (available online at http://www.regulations.gov/#!document

Detail;D=EPA-HQ-OPPT-2007-0531-0874 as of April 18, 2016)

3 Although the HPV2 test rule specifies that the toxicity to algae test should be conducted according to ASTM 

E1218-04ε1, EPA previously granted a request from Univar USA Inc. to follow OECD TG 201 instead (request 

granted in EPA’s review of the study plan submitted on April 15, 2014 for CASRN 6381-77-7, “HPV2 Review of 

Aquatic Toxicity Study Plan Dated April 15, 2014, D-erythro-Hex-2-enonic acid, gamma-lactone, monosodium salt, 

CASRN 6381-77-7”, SRC Technical Report #14-013, dated May 19, 2014). 
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C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Evaluation of Data Submitted for the Toxicity to Algae Testing Requirements:

The study submitted by Greenwich Chemical Consulting, Inc. on behalf of Univar USA Inc. in 

the submission dated March 5, 2016 is adequate to satisfy the toxicity to algae component of the 

aquatic toxicity (C1) testing requirement for CASRN 6381-77-7 promulgated in the final HPV2

test rule. 

Other HPV2 Test Rule Toxicity Testing Requirements for CASRN 6381-77-7:

Greenwich Chemical Consulting, Inc., on behalf of Univar USA Inc., previously submitted study 

reports of acute toxicity to fish1and acute toxicity to Daphnia2tests of CASRN 6381-77-7.

These studies were reviewed in a separate report3and were determined to be adequate to satisfy

the acute toxicity to fish and acute toxicity to Daphnia components of the aquatic toxicity (C1) 

testing requirement for CASRN 6381-77-7 promulgated in the final HPV2 test rule.

There are no outstanding HPV2 test rule requirements for toxicity testing of CASRN 6381-

77-7.

 

1 Sacker, D. (Study Director). 2016a. D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 

6381-77-7]: Acute toxicity to rainbow trout. Performing Laboratory: Envigo Research Limited, Derbyshire, UK. 

Sponsor: Univar USA Inc. (available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2007-0531-0878 as of April 18, 2016)

2 Sacker, D. (Study Director). 2016b. D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 

6381-77-7]: Daphnia sp., 48-hour acute immobilization test. Performing Laboratory: Envigo Research Limited, 

Derbyshire, UK. Sponsor: Univar USA Inc. (available online at http://www.regulations.gov/#!documentDetail;

D=EPA-HQ-OPPT-2007-0531-0878 as of April 18, 2016)

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“HPV2 Test Rule Data Adequacy Review: Acute Toxicity to Fish and Acute Toxicity to Daphnia, D-erythro-Hex2-enonic acid, .gamma.-lactone, monosodium salt, CASRN 6381-77-7”, SRC Technical Report #16-008, dated 

February 26, 2016. (available online at https://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-

0531-0891 as of April 18, 2016)
Final HPV2 Test Rule Data Adequacy Review (April 2016) CASRN 6381-77-7

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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 (Vryenhoef, 2016)

Title: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt [CAS RN 6381-77-7]: 

Algal growth inhibition test.

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt, CASRN 6381-77-7

Remarks: Purity 99.19%; Batch No. 14031314; Appearance/State: White, granular solid

METHOD

Method/guideline followed: OECD TG 201 (2006) “Freshwater Alga and Cyanobacteria, 

Growth Inhibition Test” referenced as Method C.3 of Commission Regulation (EC) No. 

761/2009

Test type: Toxicity to Plants – Algae (static)

GLP compliant?: Yes

Year study was performed: 2014-2016

Test species: Green algae (Pseudokirchneriella subcapitata); strain: CCAP 278/4; obtained 

from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, 

Argyll, Scotland. 

Analytical monitoring: Yes

Exposure duration: 72 hours

Concentrations tested: 

Range-finding test: 

Nominal concentrations: 0 (control), 0.10, 1.0, 10, and 100 mg/L

Definitive test: 

Nominal concentrations: 0 (control), 10, 20, 40, 80, and 160 mg/L
Final HPV2 Test Rule Data Adequacy Review (April 2016) CASRN 6381-77-7

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Geometric mean measured concentrations: < LOQ1, < LOQ, < LOQ, 1.2, 3.1, and 10 mg/L at 

nominal concentrations of 0, 10, 20, 40, 80, and 160 mg/L, respectively.

Test method/conditions remarks: Liquid cultures of Pseudokirchneriella subcapitata were 

obtained from the Culture Collection of Algae and Protozoa (CCAP). Master cultures were 

maintained in the laboratory at 21 ± 1 °C under constant aeration and constant illumination. 

“Prior to the start of the test sufficient master culture was added to approximately 100 mL 

volumes of culture media contained in conical flasks to give an initial cell density of 

approximately 103cells/mL. The flasks were plugged with polyurethane foam stoppers and kept 

under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 ± 1 °C 

until the algal cell density was approximately 104- 105cells/mL.” [Vryenhoef 2016, p. 10] 

These cultures were used to inoculate the test solutions for toxicity tests.

The range-finding and definitive tests were conducted in 250-mL glass conical flasks, each 

containing 100 mL of prepared test solution. The flasks were plugged with polyurethane foam 

bungs to reduce evaporation. The algal test medium for the range-finding and definitive tests 

was prepared using reverse osmosis purified deionized water.

Range-Finding Test Procedure: 

Freshwater algae (2 replicates/concentration; initial cell density of ~5 x 103cells/mL) were 

exposed to nominal concentrations of 0 (control), 0.10, 1.0, 10, and 100 mg/L for 72 hours. 

Preparation of test solutions:

“A nominal amount of test item (50 mg) was dissolved in culture medium and the volume 

adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made 

to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the 

stock solutions was separately inoculated with algal suspension (2.3 mL) to give the required test 

concentrations of 0.10, 1.0, 10 and 100 mg/L.” [Vryenhoef 2016, p. 11] 

“Due to the light sensitive nature of the test item, all test preparation was conducted under nonactinic lighting.” [Vryenhoef 2016, p. 11]

Evaluation of cell densities: 

Algal densities in each vessel were determined at 0 and 72 hours using a Coulter Multisizer 

Particle Counter. Mean measured cell densities at 0 hours ranged from 5.38 to 6.59 x 103

cells/mL.

Environmental conditions:

The test vessels were incubated at 24 ± 1 °C under continuous illumination (intensity ~7000 lux) 

provided by warm white lighting (380-730 nm) and were constantly shaken at approximately 150 

rpm. The study report did not provide measurements of pH or test substance concentration 

during the range-finding test.

 

1 The limit of quantification (LOQ) for the test substance was 0.94 mg/L.
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Definitive Test Procedure:

Freshwater algae (3 replicates per test concentration, 6 replicates for the control; initial cell 

density of ~5 x 103cells/mL) were exposed to nominal concentrations of 0 (control), 10, 20, 40, 

80, and 160 mg/L for 72 hours. 

Preparation of test solutions: 

“A nominal amount of test item (320 mg) was dissolved in culture medium and the volume 

adjusted to 2 liters to give a 160 mg/L stock solution from which a series of dilutions was made 

to give further stock solutions of 80, 40, 20 and 10 mg/L. An aliquot (1 liter) of each of the stock 

solutions was separately inoculated with algal suspension (7.7 mL) to give the required test 

concentrations of 10, 20, 40, 80 and 160 mg/L.” [Vryenhoef 2016, p. 13]

“Due to the light sensitive nature of the test item, all test preparation was conducted under nonactinic lighting.” [Vryenhoef 2016, p. 13]

Inoculation of test cultures: 

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

density of 6.49 x 105cells per mL. Inoculation of 1 liter of test medium with 7.7 mL of this algal 

suspension gave an initial nominal cell density of 5 x 103cells per mL and had no significant 

dilution effect on the final test concentration.” [Vryenhoef 2016, p. 13]

Evaluation of cell densities and appearance: 

Algal densities in each vessel were determined at 0, 24, 48, and 72 hours using a Coulter 

Multisizer Particle Counter. Mean measured cell densities at 0 hours ranged from 4.34 to 5.45 

x 103cells/mL. The algal cells were microscopically examined for shape and size at test 

termination.

Environmental conditions:

The test vessels were incubated at 24 ± 1 °C under continuous illumination (intensity ~7000 lux) 

provided by warm white lighting (380-730 nm) and constantly shaken at approximately 150 rpm.

The temperature in the incubator was recorded daily. The pH in each test vessel was determined 

at test initiation and after 72 hours.

Analytical measurement of test substance concentrations: 

“Samples were taken from the control and each test group from the bulk test preparation at 0 

hours and from the pooled replicates at 72 hours for immediate quantitative analysis... Two 

further samples of each test concentration were prepared at the start of the test and incubated 

under test conditions in order to provide samples for analysis at 24 and 48 hours.” [Vryenhoef 

2016, p. 14] A 5-mL aliquot of acetic acid was added to each test sample (95 mL) prior to 

analysis. Analysis of samples for test substance concentration was conducted by highperformance liquid chromatography with UV detection (wavelength: 280 nm) using a validated 

procedure. The test samples were analyzed on the day of receipt. 

Statistical analysis: 

For determination of the NOEC and LOEC values based on 72-hour growth rate and yield data, 

the data were initially checked for homogeneity of variance using Bartlett’s Test. Differences 
Final HPV2 Test Rule Data Adequacy Review (April 2016) CASRN 6381-77-7

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between treatments and the control were evaluated for statistical significance using Dunnett’s 

multiple comparison procedure. All statistical analyses were performed using the SAS computer 

software package (SAS, 1999-2001).

The study author considered the test to be valid if the following criteria were met:

 “The cell concentration of the control cultures must increase by a factor of at least 16 

over the test period. 

 The mean of the coefficients of variation of the section by section daily growth rates in 

the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) 

must not exceed 35%.

 The coefficient of variation of the average specific growth rate in replicate control 

cultures must not exceed 7%.” [Vryenhoef 2016, p. 17]

RESULTS

Endpoint value(s): 

Based on nominal concentrations:

72-hour EC50 (growth rate) > 160 mg/L

72-hour NOEC (growth rate) = 160 mg/L

72-hour EC50 (yield) > 160 mg/L

72-hour NOEC (yield) = 160 mg/L

Based on geometric mean measured concentrations:

72-hour EC50 (growth rate) > 10 mg/L

72-hour NOEC (growth rate) = 10 mg/L

72-hour EC50 (yield) > 10 mg/L

72-hour NOEC (yield) = 10 mg/L

Results remarks: 

Range-Finding Test Results: 

Cell densities and mean percentage values for inhibition of growth rate and yield relative to the 

control at each nominal test substance concentration were as follows: 

Nominal 

Concentrations

Mean Measured Cell Densities (cells 

per mL)aInhibition Values (%)

0 Hours 72 Hours Growth Rate Yield

Control (0) 5.38E+03 5.46E+05 - -

0.10 6.05E+03 5.42E+05 3 1

1.0 6.59E+03 4.43E+05 9 19

10 6.29E+03 5.40E+05 3 1

100 6.04E+03 1.90E+05 25 66

a Cell densities at 0 and 72 hours are the means of two replicates per concentration.
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“The results showed no significant effect on growth rate at the [nominal] test concentrations of 

0.10, 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L.” [Vryenhoef 

2016, p. 18] In addition, yield appeared to be reduced at 100 mg/L. 

“Based on this information test concentrations of 10, 20, 40, 80 and 160 mg/L were selected for 

the definitive test.” [Vryenhoef 2016, p. 18]

Definitive Test Results:

At 0 hours, measured test substance concentrations were < LOQ, 6.40, 17.2, 54.3, and 137 mg/L 

for the 10, 20, 40, 80, and 160 mg/L nominal test concentrations, respectively. At 72 hours, 

measured test substance concentrations were below the LOQ for all test concentrations. 

“Analysis of the test preparations at 0 hours showed measured test concentrations to range from 

less than the limit of quantification (LOQ) of the analytical method employed at [a nominal 

concentration of] 10 mg/L through to 137 mg/L at [a nominal concentration of] 160 mg/L. A 

concentration dependent decline in measured concentrations was observed after each 24-Hour 

time point [resulting in concentrations] in the range of less than the LOQ to 31 mg/L at 24 hours, 

less than the LOQ to 5.6 mg/L at 48 hours and... less than the LOQ in all test samples at 72 

hours.” [Vryenhoef 2016, p. 18]

“Given this decline in measured test concentrations it was considered justifiable to base the 

results on the geometric mean measured test concentrations in order to give a ‘worst case’ 

analysis of the data.” [Vryenhoef 2016, p. 18]

Nominal and measured concentrations (0-72 hours) and corresponding geometric mean measured 

concentrations1 were as follows:

Nominal 

Concentrations 

(mg/L)

Measured Concentrations (mg/L) Percentage 

(%) of 

0 Hours 24 Hours 48 Hours 72 Hours Nominal

Geometric 

Meana

0 (Control) < LOQ < LOQ < LOQ < LOQ < LOQ -

10 < LOQ < LOQ < LOQ < LOQ < LOQ -

20 6.40 < LOQ < LOQ < LOQ < LOQ -

40 17.2 < LOQ < LOQ < LOQ 1.2 3

80 54.3 7.42 < LOQ < LOQ 3.1 4

160 137 31.3 5.55 < LOQ 10 6

a

In cases where the measured concentration was less than the LOQ (limit of quantification) of the analytical method 

(0.94 mg/L), a value of one half of the LOQ (i.e., 0.47 mg/L) was used in the calculation of the geometric mean.

 

1 The geometric mean measured concentrations provided in this table were calculated by the reviewers using the 

following equation: Geometric mean (GM) = √C0 × C24 × C48 × C72

4 where Ct is the measured test substance 

concentration at time t (hours after initiation of exposure). In contrast, the mean measured concentrations reported 

by the study author were calculated by taking the geometric means for each 24-hour interval (i.e., √C0 × C24

2

, 

√C24 × C48

2

, and √C48 × C72

2

) and then calculating the arithmetic average of these geometric means.
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Biological results:

The 72-hour cell density in the control averaged 5.62 x 105cells/mL. Cell densities in the 10, 20, 

40, 80, and 160 mg/L nominal treatment levels averaged 8.06, 6.77, 5.61, 6.83, and 8.60 x 105

cells/mL, respectively, at 72 hours. Over the 72-hour exposure, no significant reductions in 

growth rate or yield were observed at any measured concentration, compared to control data. 

Therefore, the 72-hour NOEC value for inhibition of growth rate and yield was determined to be 

10 mg/L based on geometric mean measured concentrations. The 72-hour EC50 value was 

determined to be > 10 mg/L.

Cell densities and mean percentage values for inhibition of growth rate and yield relative to the 

control at each nominal concentration were as follows:

Nominal

Concentration 

(mg/L)

Mean Measured Cell Densities (cells 

per mL)a

Inhibition Values (%)

0 Hours 72 Hours Growth Rate Yield

0 (Control) 5.45E+03 5.62E+05 - -

10 4.34E+03 8.06E+05 [7]b[44]b

20 4.88E+03 6.77E+05 [3]b[21]b

40 5.24E+03 5.61E+05 1 0

80 4.77E+03 6.83E+05 [4]b[22]b

160 4.60E+03 8.60E+05 [8]b[54]b

a Cell densities at 0 and 72 hours are the means of six (control) or three (test substance) replicates per concentration.

b [Increase in growth rate or yield compared to controls]

Environmental conditions: 

The pH of control cultures was 7.4 at 0 hours and 8.1 at 72 hours. For treated algal cultures, pH 

ranged from 7.1 to 7.2 at 0 hours and from 8.1 to 8.2 at 72 hours. “Temperature was maintained 

at 24 ± 1 °C throughout the test.” [Vryenhoef 2016, p. 20]

Observations of the cultures: 

“All test and control cultures were inspected microscopically at 72 hours. There were no 

abnormalities detected in any of the control or test cultures.” [Vryenhoef 2016, p. 20]

Observations of test item solubility: 

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

After the 72-Hour test period all control test cultures were observed to be slightly pale green 

dispersions whilst the test cultures were observed to be green dispersions.” [Vryenhoef 2016, p. 

21]

Acceptability Criteria: 

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

 “[T]he cell concentration of the control cultures increased by a factor of 103 after 72 

hours. This increase was in line with the OECD Guideline that states the enhancement 

must be at least by a factor of 16 after 72 hours.” [Vryenhoef 2016, p. 20]
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 “The mean coefficient of variation for section by section specific growth rate for the 

control cultures was 16% and hence satisfied the validation criterion given in the OECD 

Guideline which states the mean must not exceed 35%.” [Vryenhoef 2015, p. 20]

 “The coefficient of variation for average specific growth rate for the control cultures over 

the test period (0 - 72 h) was 2% and hence satisfied the validation criterion given in the 

OECD Guideline which states that this must not exceed 7%.” [Vryenhoef 2015, p. 20]

CONCLUSIONS

No effects on algal growth or yield were observed up to a nominal test concentration of 160 

mg/L (geometric mean measured concentration of 10 mg/L). Based on geometric mean 

measured concentrations1, the 72-hour NOEC value was 10 mg/L for inhibition of growth rate 

and yield. The 72-hour EC50 value was > 10 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. In the range-finding test, inhibition of growth rate (25%) and yield 

(66%) were observed in algal cultures exposed to a nominal test substance concentration of 100 

mg/L, relative to the control group. However, no effects on growth rate or yield were observed 

under similar conditions in the definitive test at nominal concentrations up to 160 mg/L, which 

corresponded to a geometric mean measured concentration of 10 mg/L. Test concentrations 

were not analyzed in the range-finding test; therefore, the amount of test substance present in the 

test vessels during the range-finding test is unknown. The study author did not address the 

discrepancy in toxicity test results between the range-finding and definitive tests. In addition, the 

reviewers disagree with the methods used by the study author to calculate the mean measured 

test substance concentrations. The mean measured concentrations provided in this summary 

were calculated by the reviewers as the geometric means of the four measurements (i.e., at 0, 24, 

48, and 72 hours) reported for each treatment level during the study.

REFERENCE

Vryenhoef, H. (Study Director). 2016. D-erythro-Hex-2-enonic acid, .gamma.-lactone, 

monosodium salt [CAS RN 6381-77-7]: Algal growth inhibition test. Performing Laboratory: 

Envigo Research Limited, Derbyshire, UK. Study No. 41401175. Sponsor: Univar USA Inc.

Text in quotes is taken directly from Vryenhoef (2016).

 

1 Compared with test substance concentrations measured at 0 hours in the definitive study, there was a significant 

decline in the measured concentrations at 24, 48, and 72 hours; therefore, the study author stated that “it was 

considered justifiable to base the results on the geometric mean measured test concentrations in order to give a 

‘worst case’ analysis of the data.” [Vryenhoef 2015, p. 18]