SRC TR-16-008

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL): 

ACUTE TOXICITY TO FISH AND ACUTE TOXICITY TO DAPHNIA

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

Prepared by:

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

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

February 26, 2016
Final HPV2 Test Rule Data Adequacy Review (February 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. Acute Toxicity to Fish ....................................................................................................... 3

D-2. Acute Toxicity to Aquatic Invertebrates............................................................................ 9
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates acute toxicity to fish and acute toxicity to Daphnia studies of CASRN 

6381-77-7 (D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt; sodium 

erythorbate) submitted on January 15, 2016 by Greenwich Chemical Consulting, Inc. (on behalf 

of Univar USA 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 test rule; available online at http://www.regulations.gov/#!document

Detail;D=EPA-HQ-OPPT-2007-0531-0579 as of February 26, 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 < -1.551, 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 January 15, 2016 submission from Greenwich Chemical Consulting, Inc. (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0878 as of 

February 26, 2016) contained the following study reports: 

 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.

 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.

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. Each 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.

2

 If a study was 

determined to be inadequate, a basis for that conclusion is supplied.

 

1 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 February 26, 2016)

2 Although the HPV2 test rule specifies that acute toxicity to fish and acute toxicity to Daphnia tests should be 

conducted according to ASTM E729-96, EPA previously granted a request from Univar USA Inc. to follow OECD 

TG 203 and OECD TG 202, respectively, 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-
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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

Evaluation of Data Submitted for the Acute Toxicity to Fish and Acute Toxicity to Daphnia

Testing Requirements:

The studies provided in the January 15, 2016 submission and reviewed in this document are 

considered 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.

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

Data have not yet been submitted to EPA for the toxicity to algae component of the aquatic 

toxicity (C1) testing requirement for CASRN 6381-77-7.

Summary of Data Adequacy for Toxicological Endpoints:

Available data are adequate to satisfy the testing requirements for the acute toxicity to fish 

and acute toxicity to Daphnia endpoints. The toxicity to algae endpoint is considered to be 

a data gap and testing is still required for this endpoint.

 

erythro-Hex-2-enonic acid, gamma-lactone, monosodium salt, CASRN 6381-77-7”, SRC Technical Report #14-013, 

dated May 19, 2014). 
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Oncorhynchus mykiss (Sacker, 2016a)

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

7]: Acute toxicity to rainbow trout

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 203 (1992) and Method C.1 of Commission Regulation 

(EC) No. 440/2008

Test type: Acute Toxicity to Fish – Flow-through conditions

GLP compliant?: Yes

Year study performed: 2015

Test species: Rainbow trout (Oncorhynchus mykiss)

Analytical monitoring: Yes 

Exposure duration: 96 hours

Concentrations tested: 

Range-finding test: 

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

Geometric mean measured concentrations1: 5.36 and 96.2 mg/L at nominal concentrations of 10 

and 100 mg/L, respectively

Definitive test: 

Nominal concentrations: 0 (control) and 100 mg/L

 

1 Geometric mean measured concentrations of the test substance were calculated by the reviewers.
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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Geometric mean measured concentrations1: <LOQ2and 76.1 mg/L at nominal concentrations of 

0 and 100 mg/L, respectively

Test method/conditions remarks:

Test species: 

Juvenile rainbow trout (Oncorhynchus mykiss) were obtained from Brow Well Fisheries Limited, 

Hebden, UK and maintained in-house. The fish were acclimated to dilution water and test 

conditions for approximately 12 days prior to testing. During the acclimation period, the lighting 

cycle was 16 hours light/8 hours darkness with dawn and dusk transition periods of 20 minutes. 

“There was no mortality in the 7 days prior to the start of the test and the fish had a mean 

standard length of 4.0 cm (sd = 0.3) and a mean weight of 0.88 g (sd = 0.15) at the end of the 

definitive test. Based on the mean weight value this gave a loading rate of 0.07 g of 

bodyweight/liter [per 24 hours] ([calculated using a flow-through] volume in 24 hours = 86.4 

liters).” [Sacker 2016a, p. 10] 

Preparation of test solutions: 

The dilution water was laboratory tap water that had been dechlorinated by filtration through 

activated carbon and partially softened to achieve an approximate hardness of 140 mg/L as 

CaCO3. After filtration and softening, water was heated via a series of computer-controlled plate 

heat exchangers to the required temperature for the toxicity test. Analysis of the dilution water, 

conducted prior to and after testing (in July 2015 and January 2016)3, showed “total water 

hardness of 134 and 136 mg/L as CaCO3, alkalinity of 116 and 78.8 mg/L as CaCO3, [and] Total 

Organic Carbon content of 0.36 and 3.1 mg C/L”, respectively. [Sacker 2016a, p. 11] 

“Due to the potentially light sensitive nature of the test item, all test item preparation was 

performed under non-actinic lighting/shielded from the light.” [Sacker 2016a, p. 11]

Range-finding test: 

The limit test concentration selected for the definitive test was based upon the results of a single 

96-hour preliminary range-finding test. In this preliminary test, groups of three fish per 

concentration were exposed to nominal concentrations of 0 (control), 10, or 100 mg/L for 96 

hours under flow-through conditions. 

“Nominal amounts of test item (1.2 and 12 g) were dissolved in deionized reverse osmosis water 

and the volume adjusted to 1 liter to give the 1.2 and 12 g/L stock solution respectively. The 

stock solutions were sonicated for 10 minutes and inverted several times to ensure adequate 

mixing and homogeneity. Dilution water was dosed at a flow rate of 60 mL per minute using [a] 

Watson Marlow 505S peristaltic pump and the stock solutions at a flow rate of 0.50 mL per 

minute using a Watson Marlow 205U peristaltic pump. The dilution water and stock solutions 

were combined in separate mixing vessels and delivered to the test vessels to give the required 

test concentrations of 10 and 100 mg/L.” [Sacker 2016a, p. 11]

 

1 Geometric mean measured concentrations of the test substance were calculated by the reviewers.

2 The limit of quantitation (LOQ) for the test substance was 2.6 mg/L.

3 The acute toxicity to fish study was conducted from September 22, 2015 to October 30, 2015.
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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Each group of three fish was placed in a 25-L test vessel, which contained 20 L of test media and 

was covered to reduce evaporation. Test vessels were kept in a 14 °C temperature-controlled 

room in the dark. Fish were inspected for sublethal effects and mortalities at 3, 6, 24, 48, 72, and 

96 hours after the start of the test. Samples of test media were extracted from each test vessel at 

0, 72, and 96 hours for analysis of test substance concentrations. 

Definitive test: 

The definitive test was conducted as a limit test. Groups of seven fish per concentration were 

exposed to nominal concentrations of 0 (control) or 100 mg/L for 96 hours under flow-through 

conditions.

“A nominal amount of test item (12 g) was dissolved in deionized reverse osmosis water and the 

volume adjusted to 1 liter to give the 12 g/L stock solution. The stock solution was sonicated for 

5 minutes and inverted several times to ensure adequate mixing and homogeneity. Dilution 

water was dosed at a flow rate of 60 mL per minute using [a] Watson Marlow 505S peristaltic 

pump and the stock solution at a flow rate of 0.50 mL per minute using a Watson Marlow 205U 

peristaltic pump. The dilution water and stock solution were combined in a mixing vessel and 

delivered to the test vessel to give the required test concentration of 100 mg/L.” [Sacker 2016a, 

p. 12]

“Continuous flow-through test conditions were employed in the test in an effort to maintain 

dissolved test item concentrations. As in the range-finding test, 25 liter glass exposure vessels 

containing 20 liters of test media were used for each control and test concentration. At the start 

of the test seven fish were placed in each test vessel at random, in the test preparations. The test 

vessels were then covered to reduce evaporation and maintained at 14 °C in a temperature 

controlled room in the dark for a period of 96 hours. The test solutions were not aerated. The 

fish were not individually identified and received no food during exposure.” [Sacker 2016a, p. 

12]

The fish were observed for mortality or sublethal effects at approximately 3, 6, 24, 48, 72 and 96 

hours after the start of exposure. Dissolved oxygen, pH, and temperature data were measured 

daily throughout the duration of the test. 

“Water samples were taken from the control and 100 mg/L test vessel at 0, 24, 48, 72, and 96 

hours for quantitative analysis.” [Sacker 2016a, p. 13] A second sample was removed from each 

flask and stored frozen for possible further analysis. 

Analytical measurement of test substance concentrations:

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 high performance liquid 

chromatography with UV detection (HPLC/UV). The test samples were analyzed on the day of 

receipt. “The analytical methodology was not validated prior to the analysis of range-finding 

samples; full validation was later conducted at a suitable concentration.” [Sacker 2016a, p. 30] 
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Statistical analysis: 

No statistical analyses or methods were used for this test. The LC50 value was estimated based 

on mortality data.

Criteria for validity of the study: 

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

 “In the control, not more than one of the fish should die or show signs of stress during 

the 96 hours. 

 The dissolved oxygen concentration at the end of the test should be greater than or 

equal to 60% of Air Saturation Values (equivalent to 6.1 mg O2/L) in the control and 

test vessels.” [Sacker 2016a, p. 13]

RESULTS

Endpoint value(s): 

Based on nominal concentrations:

96-hour NOEC = 100 mg/L

96-hour LC50 > 100 mg/L

Based on geometric mean measured concentrations:

96-hour NOEC = 76.1 mg/L

96-hour LC50 > 76.1 mg/L

Results remarks: 

Range-finding test:

After 96 hours, no fish mortality was observed at any concentration. However, a sublethal effect 

was observed at 100 mg/L; at this concentration, one fish was swimming on its side at 96 hours. 

The study author noted that this fish had physical damage to its tail, which was believed to have 

resulted from aggressive behavior of other fish, rather than from toxicity. Therefore, the study 

author did not consider the sublethal effect to be related to exposure to the test substance. Based 

upon these results, a single nominal test concentration of 100 mg/L was selected for the 

definitive test. 

Chemical analysis of the test preparations at 0, 72, and 96 hours showed measured test 

concentrations ranging from 32 to 103% of nominal concentrations. At the nominal

concentration of 10 mg/L, measured concentrations were 32 to 75% of nominal. At the nominal 

concentration of 100 mg/L, measured concentrations were 91 to 103% of nominal. The 

geometric mean measured concentrations were 5.36 and 96.2 mg/L at nominal concentrations of 

10 and 100 mg/L, respectively. No analytical data were provided for the control.

Definitive test:

No mortalities or sublethal effects were observed in the control or limit test group during the 96-

hour exposure. 

HPLC/UV analysis of the test preparations at 24, 48, 72 and 96 hours showed measured test 

concentrations ranging from 80 to 96% of nominal concentrations. The analysis at 0 hours 
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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showed measured test concentrations ranging from 11 to 48% of nominal concentrations. 

Measured concentrations of the test solutions are provided in the table below: 

Measured Concentrations (mg/L)

Nominal 

Concentrations 

(mg/L) 0 hours 24 hours 48 hours 72 hours 96 hours

Geometric 

mean

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

100 47.7/10.8a82.7 83.9 79.9 96.4 76.1

a This was a duplicate sample, which was stored frozen prior to analysis. 

LOQ = Limit of quantitation (2.6 mg/L)

The study author suspected the analytical measurement of 47.7 mg/L at 0 hours to be anomalous. 

However, the study author did not provide evidence to demonstrate that this measurement was 

not representative of actual concentrations. Therefore, the EPA reviewers included this 

measurement in their calculation of the geometric mean measured concentration. The 

measurement of 10.8 mg/L, which was obtained from a duplicate sample that had been stored 

frozen prior to analysis, was not included in the calculation of the geometric mean measured 

concentration because it appeared to be the only sample that was not analyzed directly after 

sampling occurred and the measurement was remarkably different from all other measurements.

The test solutions were observed to be clear and colorless at the start and throughout the test. 

During the 96-hour exposure, dissolved oxygen levels ranged from 8.6 to 9.6 mg/L, temperature 

was maintained at 14 °C, and pH ranged from 7.4 to 7.5. 

According to the study author, “[t]he test was considered to be valid given that none of the 

control fish died or showed signs of stress during the test and that the oxygen concentration at 

the end of the test was greater than or equal to 60% of ASV (6.1 mg O2/L) in the control and test 

vessels.” [Sacker 2016a, p. 15] 

CONCLUSIONS

No mortality and no signs of toxicity were observed at the limit test concentration (nominal 

concentration = 100 mg/L; geometric mean measured concentration = 76.1 mg/L). Based on the 

nominal concentration of the test substance, the 96-hour LC50 was > 100 mg/L in Oncorhynchus 

mykiss. Based on the geometric mean measured concentration of the test substance, the 96-hour 

LC50 was > 76.1 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions. The mean measured concentration of the limit test was 76.1 mg/L, 

rather than the concentration of 100 mg/L specified by OECD TG 203 for a limit test. However, 
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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because the test substance appears to be unstable under standard laboratory conditions (e.g., 

susceptible to photolytic degradation)1, the achieved concentration of 76.1 mg/L is acceptable.

REFERENCE

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.

Text in quotes was taken directly from the report of Sacker (2016a).

 

1

In an amendment to the acute toxicity to fish protocol (submitted via email to Mr. John Schaeffer of EPA from Mr. 

John V. Massingale of Greenwich Chemical Consulting, Inc., on behalf of Univar USA, Inc. on June 2, 2015), 

Harlan Laboratories reported that the test substance was unstable in fish toxicity test medium under standard test 

conditions, based on the loss of 95% of the test substance (i.e., CASRN 6381-77-7) after 24 hours in stability trials 

under static conditions with standard laboratory lighting.
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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D-2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (Sacker, 2016b)

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

7]: Daphnia sp., 48-hour acute immobilization 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 202 (2004) and Method C.2 of Commission Regulation 

(EC) No. 440/2008

Test type: Acute toxicity to Daphnia – Flow-through conditions

GLP compliant?: Yes

Year study performed: 2014-2015

Test species: Water flea (Daphnia magna) neonates (< 24 hours old), 1st instar derived from inhouse cultures

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested: 

Range-finding test: 

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

Geometric mean measured concentrations1: 8.41 and 95.3 mg/L at nominal concentrations of 10 

and 100 mg/L, respectively

Definitive test: 

Nominal concentrations: 0 (control) and 100 mg/L

Geometric mean measured concentrations1: < LOQ2and 91.5 mg/L at nominal concentrations of 

 

1 Geometric mean measured concentrations of the test substance were calculated by the reviewers. 

2 The limit of quantitation (LOQ) for the test substance was 2.4 mg/L.
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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0 and 100 mg/L, respectively

Test method/conditions remarks: 

Test species:

The study was performed with the freshwater daphnid, Daphnia magna, from in-house cultures 

maintained in Elendt M7 medium. Test organisms were young daphnids, which were less than 

24 hours old and were in the 1st instar. 

Preparation of test solutions: 

For both the range-finding and definitive tests, reconstituted water (ISO medium) was used as 

the dilution water during exposure. The prepared reconstituted water had a pH of 7.8±0.2, an 

approximate theoretical total hardness of 250 mg CaCO3/L, and a dissolved oxygen 

concentration that approximated the air saturation value.

All test preparations were conducted under non-actinic lighting (or shielded from the light) due 

to the potentially light-sensitive nature of the test substance.

Range-finding test: 

The limit test concentration selected for the definitive test was based upon the results of a single 

48-hour preliminary range-finding test. In this preliminary test, groups of 10 daphnids per 

concentration were exposed to nominal concentrations of 0 (control), 10, or 100 mg/L for 48 

hours under flow-through conditions. 

“Nominal amounts of test item (1.2 and 12 g) were dissolved in deionized reverse osmosis water 

and the volume adjusted to 1 liter to give the 1.2 and 12 g/L stock concentrations. The stock 

solutions were sonicated for 5 minutes and inverted several times to ensure adequate mixing and 

homogeneity. Dilution water was dosed at a flow rate of 30 mL per minute using [a] Watson 

Marlow 505S peristaltic pump and the stock solutions at a flow rate of 0.25 mL per minute using 

a Watson Marlow 205U peristaltic pump. The dilution water and stock solutions were combined 

in separate mixing vessels and delivered to the test vessels to give the required test 

concentrations of 10 and 100 mg/L.” [Sacker 2016b, p. 11]

Each group of 10 daphnids was contained in a 150-mL test vessel (glass beaker with stainless 

steel mesh sides) that was placed in a tank containing ~ 5 L of test solution. The test was 

conducted in the dark in a temperature-controlled room at approximately 20 °C. Daphnids were 

observed for immobilization (inability to swim within 15 seconds after gentle agitation of the test 

vessel) and other adverse reactions at 24 and 48 hours after the start of exposure. 

A sample of test medium was extracted from each test vessel at 0 and 48 hours for analysis of 

test substance concentrations. In order to stabilize the test item, a 5-mL aliquot of glacial acetic 

acid was added to each sample bottle before addition of 95 mL of the test sample.
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Definitive test: 

The definitive test was conducted as a limit test. Four groups of five daphnids per concentration 

were exposed to nominal concentrations of 0 (control) or 100 mg/L for 48 hours under flowthrough conditions.

“A nominal amount of test item (12 g) was dissolved in deionized reverse osmosis water and the 

volume adjusted to 1 liter to give the 12 g/L stock solution. The stock solution was sonicated for 

5 minutes and inverted several times to ensure adequate mixing and homogeneity. Dilution 

water was dosed at a flow rate of 30 mL per minute using [a] Watson Marlow 505S peristaltic 

pump and the stock solution at a flow rate of 0.25 mL per minute using a Watson Marlow 205U 

peristaltic pump. The dilution water and stock solution were combined in a mixing vessel and 

delivered to the test vessels to give the required test concentration of 100 mg/L.” [Sacker 2016b, 

p. 12]

A fresh stock solution was prepared daily and the flow rates were adjusted as necessary.

“Continuous flow-through test conditions were employed in the test in an effort to maintain 

dissolved test item concentrations. As in the range-finding test glass test vessels (150 mL 

beakers) with stainless steel mesh sides were placed in a tank retaining approximately 5 liters of 

test solution. At the start of the test 5 daphnids were placed in each test and control vessel at 

random, in the test preparations. Four replicate test and control vessels were prepared. The test 

vessels were then covered to reduce evaporation and maintained in a temperature controlled 

room at approximately 20 °C in the dark. The daphnids were not individually identified, 

received no food during exposure and the test vessels were not aerated.” [Sacker 2016b, p. 12] 

Daphnids were observed for immobilization or other adverse reactions at 24 and 48 hours after 

the start of exposure. Dissolved oxygen and pH were measured at 0 and 48 hours. Water 

temperature was measured daily during exposure. 

“Samples were taken from the control and the 100 mg/L test group for analysis every 24 hours 

throughout the test. Duplicate samples were taken at each occasion and stored frozen for further 

analysis if necessary. In order to ensure stability of the test item, a volume of glacial acetic acid 

(5 mL) was added to each sample bottle before addition of [95 mL of] the test sample.” [Sacker 

2016b, p. 13]

Analytical measurement of test substance concentrations:

Analysis of samples for test substance concentration was conducted by high performance liquid 

chromatography with UV detection (HPLC/UV). The test samples were analyzed on the day of 

receipt. “The analytical methodology was not validated prior to the analysis of range-finding 

samples; full validation was later conducted at a suitable concentration.” [Sacker 2016b, p. 29] 

Statistical analysis: “An estimate of the EC50 values was given by inspection of the 

immobilization data. The No Observed Effect Concentration (NOEC) at 24 and 48 hours was 

calculated using the Fisher's Exact Binomial Test with Bonferroni correction using the ToxRat 

Professional computer software package (TOXRAT).” [Sacker 2016b, p. 13] 
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Criteria for validity of the study: 

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

 “No more than 10% of the control daphnids show immobilization or other signs of 

disease or stress (e.g. discoloration or unusual behavior such as trapping at the surface 

water). 

 The dissolved oxygen concentration at the end of the test is equal to or greater than 3 

mg/L in the control and test vessels.” [Sacker 2016b, p. 13]

RESULTS

Endpoint value(s):

Based on nominal concentrations:

48-hour NOEC = 100 mg/L

48-hour EC50 > 100 mg/L

Based on geometric mean measured concentrations:

48-hour NOEC = 91.5 mg/L

48-hour EC50 > 91.5 mg/L

Results remarks: 

Range-finding test: 

After 48 hours, no immobilization or other signs of toxicity were observed at any test 

concentration. Based upon this information, a single nominal test concentration of 100 mg/L 

was selected for the definitive test. 

Chemical analysis of the test preparations showed measured concentrations at 0 hours to range 

from 79 to 92% of nominal concentrations and measured concentrations at 48 hours to range 

from 89 to 98% of nominal concentrations. Therefore, the test item was considered to be 

relatively stable under test conditions. The geometric mean measured concentrations were 8.41 

and 95.3 mg/L at nominal concentrations of 10 and 100 mg/L, respectively. No analytical data 

were provided for the control.

Definitive test: 

At 48 hours, cumulative percent immobility at nominal test concentrations of 0 (control) and 100 

mg/L were 10% and 20%, respectively. However, the percent immobilization at the limit test 

concentration was not statistically significantly different from that of the control group. Other 

adverse effects were also observed in the control and test groups. In the control group, one 

daphnid was trapped at the surface at 24 hours, but appeared normal at 48 hours. At 100 mg/L, 

reduced mobility was observed in two daphnids at 24 and 48 hours and one daphnid was covered 

in debris at 48 hours. 

HPLC/UV analysis of the test preparations at 0, 24, and 48 hours showed measured test 

concentrations ranging from 89 to 94% of nominal concentrations. Measured concentrations of 

the test solutions are provided in the table below: 
Final HPV2 Test Rule Data Adequacy Review (February 2016) CASRN 6381-77-7

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Measured Concentrations (mg/L)

Nominal 

Concentrations 

(mg/L) 0 hours 24 hours 48 hours

Geometric 

mean

Percent 

Immobilization

(%)

0 (Control) < LOQ < LOQ < LOQ < LOQ 10

100 93.5 92.5 88.5 91.5 20

LOQ = Limit of quantitation (2.4 mg/L)

The test solutions were observed to be clear and colorless at the start and throughout the test. 

During the 48-hour exposure, dissolved oxygen levels ranged from 8.4 to 8.9 mg/L, temperature 

ranged from 21 to 22 °C, and pH ranged from 8.0 to 8.1. 

According to the study author, “[t]he test was considered to be valid given that no more than 

10% of the control daphnids showed immobilization or other signs of disease or stress and that 

the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control 

and test vessels.” [Sacker 2016b, p. 15] 

CONCLUSIONS

No statistically significant increase in immobilization was observed at the limit test concentration 

(nominal concentration = 100 mg/L; geometric mean measured concentration = 91.5 mg/L), 

compared to the control. Based on the nominal concentration of the test substance, the 48-hour 

EC50 was > 100 mg/L in Daphnia magna. Based on the geometric mean measured concentration 

of the test substance, the 48-hour EC50 was > 91.5 mg/L. 

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

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.

Text in quotes was taken directly from the report of Sacker (2016b).