SRC TR-15-050

HPV2 TEST RULE DATA ADEQUACY REVIEW (FINAL):

ACUTE TOXICITY TO FISH AND ACUTE TOXICITY TO DAPHNIA

D-gluco-heptonic acid, monosodium salt, (2.xi.)-, CASRN 31138-65-5

Prepared by:

Justine von Runnen, William 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 19, 2015
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TABLE OF CONTENTS

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

B. EVALUATING DATA ADEQUACY ..................................................................................... 2

C. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 2

D. DATA FOR SIDS ENDPOINTS.............................................................................................. 4

D-1. Acute Toxicity to Fish ....................................................................................................... 4

D-2. Acute Toxicity to Aquatic Invertebrates............................................................................ 9

E. DATA ADEQUACY SUMMARY TABLE........................................................................... 14
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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates two aquatic toxicity tests for D-gluco-heptonic acid, monosodium salt, 

(2.xi.)- (CASRN 31138-65-5) submitted by Harcros Chemicals, Inc. (on May 28, 2015 and June 

22, 2015) 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-OPPT2007-0531-0579 as of August 19, 2015). 

The HPV2 test rule requires testing of CASRN 31138-65-5 for the following toxicological 

endpoints: C1 (aquatic toxicity, consisting of Test Group 1 [acute toxicity to fish, acute toxicity 

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

Daphnia and toxicity to algae tests] if log Kow ≥ 4.2), D (acute mammalian toxicity test), E1 

(bacterial reverse mutation test), E2 (chromosomal aberration or micronucleus test), and F1 

(combined repeated dose toxicity study with the reproduction/developmental toxicity screening 

test). Based on an estimated log Kow of -6.441

, Test Group 1 testing is required to determine the 

toxicity of CASRN 31138-65-5 to aquatic organisms. 

Harcros Chemicals, Inc. previously submitted acute toxicity to fish and acute toxicity to Daphnia

studies to address components of the C1 testing requirement promulgated in the final HPV2 test 

rule. These studies were reviewed by EPA2 and determined to be inadequate to satisfy the acute 

toxicity to fish and acute toxicity to Daphnia testing requirements primarily because the pH of 

the test solution preparations was adjusted to 7 prior to addition of the test organisms; this 

neutralization of the pH may have altered the toxicity of the test solutions to fish and Daphnia. 

In July 2014, Harcros Chemicals, Inc. submitted study plans to EPA for two aquatic toxicity 

tests3,4 with CASRN 31138-65-5. In response to EPA’s study plan review (SRC Technical 

Report #14-030), Harcros Chemicals, Inc. submitted revised study plans to EPA on October 13, 

20145

. EPA reviewed the revised study plans and provided comments and recommendations for 

conducting adequate studies for the acute toxicity to fish and acute toxicity to Daphnia endpoints

(SRC Technical Report #14-049).

In May and June of 2015, Harcros Chemicals, Inc. submitted the following studies to EPA to 

address components of the C1 testing requirement:

 1 U.S. EPA. 2012. Estimation Programs Interface SuiteTM (EPI Suite) for Microsoft® Windows, v4.11. U.S. 

Environmental Protection Agency, Washington, DC, USA. Available online at http://www.epa.gov/opptintr/

exposure/pubs/episuitedl.htm as of August 19, 2015. 2 Detailed reviews of previously submitted studies for the acute toxicity to fish and Daphnia endpoints are available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0807 and 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0806, respectively, as of August 19, 

2015. 3 Harris, S. 2014a. Study plan, Sodium glucoheptonate: Acute toxicity to rainbow trout. Test Facility: Harlan 

Laboratories Ltd. Study No. 41402585. Sponsor: Harcros Chemicals, Inc. Submitted to EPA in July 2014.

4 Harris, S. 2014b. Study plan, Sodium glucoheptonate: Daphnia sp., 48-hour acute immobilization test. Test 

Facility: Harlan Laboratories Ltd. Study No. 41402586. Sponsor: Harcros Chemicals, Inc. Submitted to EPA in 

July 2014.

5 Revised study plans for acute toxicity to fish and Daphnia tests are available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0825 as of August 19, 2015.
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• Acute Toxicity to Rainbow Trout, submitted to EPA on June 22, 2015 

• Acute Toxicity to Daphnia, submitted to EPA on May 28, 2015 (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0863 as of 

August 19, 2015)

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

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

Newly submitted data for the acute toxicity to fish and acute toxicity to Daphnia endpoints 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 current standard test guidelines to establish 

whether each study met current testing requirements. If the data available for the endpoint were 

determined to be inadequate, a basis for that conclusion is supplied. A table summarizing data 

availability/adequacy for each SIDS endpoint is included in Section E of this document.

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 on May 28, 2015 and June 22, 2015:

Data supplied in the May and June 2015 submissions are adequate to satisfy the acute toxicity to 

fish (component of C1) and acute toxicity to Daphnia (component of C1) testing requirements 

promulgated in the final HPV2 test rule.

Remaining HPV2 Test Rule Toxicity Testing Requirements:

In several previous submissions (dated from December 18, 2012 to August 27, 2013), Harcros 

Chemicals, Inc. submitted data for toxicity to plants (algae) (component of C1), acute 

mammalian toxicity (D), gene mutation (E1), chromosomal aberrations (E2), and combined 

repeated-dose toxicity with reproduction/developmental toxicity screening (F1) testing 

requirements promulgated in the final HPV2 test rule. These submissions included the 

following:

• Algal Inhibition Test, submitted for C1 testing requirement on April 23, 2013 (available 

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

0716 as of August 19, 2015)

• Acute Toxicity in the Rat – Up and Down Procedure, submitted for D testing requirement 

on March 19, 2013 (available online at http://www.regulations.gov/#!documentDetail;

D=EPA-HQ-OPPT-2007-0531-0710 as of August 19, 2015) 

• Reverse Mutation Assay “Ames test” using Salmonella typhimurium and Escherichia 

coli, submitted for E1 testing requirement on December 18, 2012 (available online at 
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http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0688 as of 

August 19, 2015)1

• Micronucleus Test in the Mouse, submitted for E2 testing requirement on March 7, 2013 

(available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2007-0531-0709 as of August 19, 2015)

• Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental 

Toxicity Screening Test in the Rat (OECD 422), submitted for F1 testing requirement on 

August 27, 2013 (available online at http://www.regulations.gov/#!documentDetail;D=

EPA-HQ-OPPT-2007-0531-0790 as of August 19, 2015)

The data from the submissions above have been reviewed in separate documents (SRC Technical 

Reports #13-045 and #13-071). The data were determined to be adequate to satisfy the toxicity 

to plants (algae) (component of C1), acute mammalian toxicity (D), gene mutation (E1), 

chromosomal aberrations (E2), and combined repeated-dose toxicity with 

reproduction/developmental toxicity screening (F1) testing requirements. 

Summary of Data Adequacy for Toxicological Endpoints:

Adequate data have been submitted by Harcros Chemicals, Inc. for acute toxicity to fish, acute 

toxicity to Daphnia, toxicity to plants (algae), acute mammalian toxicity, repeated-dose toxicity, 

gene mutation, chromosomal aberrations, reproductive toxicity, and developmental toxicity 

endpoints.

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

31138-65-5.

 1 Harcros Chemical, Inc. submitted an amendment to this study report on May 22, 2013 (available online at 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2007-0531-0718 as of August 19, 2015) that 

indicated that formulated concentrations used in Experiments 1 and 2 were adjusted for water content (49.5%).
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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: 96-Hour acute toxicity study in rainbow trout (Harris, 2015a) 

Title: Sodium glucoheptonate CAS No. 31138-65-5: Acute toxicity to rainbow trout 

This study is: Adequate Not Adequate

 

TEST SUBSTANCE 

Identity: D-gluco-heptonic acid, monosodium salt, (2.xi.)- (synonym: sodium glucoheptonate); 

CASRN 31138-65-5

Remarks: Amber-colored liquid; Batch No. T-461; Purity: 39.94%; 

Composition:

25.78% Beta Sodium Glucoheptonate (CASRN 30090-50-31)

14.16% Alpha Sodium Glucoheptonate (CASRN 13007-85-7)

10.96% Polysaccharides or Degradation Products

0.3 ppm Cyanide

1113 ppm Ammonia

49.1% Water

pH = 9.1

Specific Gravity = 1.293

% Solids = 50.9%

No dextrose detected 

Based on the compositional information provided, the purity of the test substance was 39.94% 

a.i.2 (25.78% beta sodium glucoheptonate + 14.16% alpha sodium glucoheptonate).

METHOD

Method/guideline followed: OECD TG 203 (1992; “Fish, Acute Toxicity Test”), Method C.1 

of Commission Regulation (EC) No. 440/2008, and OPPTS 850.1075 (1996; “Fish Acute 

Toxicity Test, Freshwater and Marine”) 

Test type: Acute Fish Toxicity Test (Semi-Static)

GLP compliant?: Yes

 1 30090-50-3 is not a valid CAS number according to the U.S. National Library of Medicine’s ChemIDplus website 

(http://chem.sis.nlm.nih.gov/chemidplus/); however, based on the chemical name provided (beta sodium 

glucoheptonate), this component is assumed to be a stereoisomer of CASRN 31138-65-5. 2 Active ingredient (a.i.) = CASRN 31138-65-5
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Year study was performed: 2015

Test species: Rainbow trout (Oncorhynchus mykiss) obtained from Brow Well Fisheries 

Limited, located in Hebden, near Skipton, Yorkshire, UK. Fish were acclimatized to test 

conditions for 7 days prior to exposure. The definitive test fish were juveniles that had a mean 

standard length of 3.9 cm and a mean weight of 0.72 g at the end of the definitive test. The 

loading rate was reported to be 0.25 g bodyweight/L.

Analytical monitoring: Yes

Exposure duration: 96 hours 

Concentrations tested: Nominal: 0 and 100 mg a.i./L (definitive and range-finding tests)

Test method/conditions remarks:

Dilution Water:

Dechlorinated laboratory tap water was partly softened to achieve a total hardness of 

approximately 140 mg CaCO3/L. At the start of the definitive test, the dilution water had a 

particulate matter concentration of 0.1 mg/L, total water hardness of 134 mg CaCO3/L, alkalinity 

of 112.4 mg CaCO3/L, Total Organic Carbon (TOC) content of 2.0 mg C/L, chemical oxygen 

demand of 1.36 mg O2/L, conductivity of 297 μS/cm, and chlorine content ranging from 0.008 to 

0.03 mg Cl2/L.

Photoperiod:

For both the range-finding and definitive tests, the photoperiod was 16 hours light and

8 hours darkness with 20 minute dawn and dusk transition periods.

Range-Finding Test Procedure: 

In the range-finding test, three fish were placed in a test vessel containing the test substance at a 

nominal concentration of 100 mg a.i./L and exposed for 96 hours under static conditions. A 

control group of three fish was maintained under identical conditions but not exposed to the test 

substance. The rationale for selection of the nominal concentration of 100 mg a.i./L was based 

on data obtained from a Daphnia magna study (Harlan Study No. 41402586; reviewed under 

section D-2, “Acute Toxicity to Aquatic Invertebrates” of this report), which suggested that 

toxicity to aquatic organisms would not be expected at this concentration.

Test Conditions:

Test vessels (25- to 30-L glass exposure vessels) contained 20 L of test solution and were 

covered to reduce evaporation. Test vessels were maintained at approximately 14°C in a 

temperature-controlled room. 

Test Solution Preparation:

To prepare the 100 mg a.i./L test solution (which was corrected for a purity of 39.94%), a 

nominal amount of test substance (5007.5 mg) was dissolved in dilution water and the volume 
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adjusted to 20 L. The solution was then stirred using a flat-bladed mixer for approximately 1 

minute to ensure homogeneity. 

Analysis of Test Solutions:

The stability and concentration of the test substance in the 100 mg a.i./L solution was verified by 

chemical analysis (HPLC-MS) at 0 and 24 hours. “The analytical methodology was not 

validated prior to the analysis of range-finding samples; full validation was later conducted 

specifically at the concentration employed in the definitive test.” [Harris 2015a, p. 33]

Definitive Test Procedure: 

Based on the results of the range-finding test, a limit test was conducted at a concentration of 100 

mg a.i./L. In the limit test, ten fish were placed in a test vessel containing the test substance at a 

nominal concentration of 100 mg a.i./L and exposed for 96 hours under semi-static conditions 

with daily renewal. A control group of ten fish was maintained under identical conditions but 

not exposed to the test substance. 

Test Conditions:

Test vessels (25- to 30-L glass exposure vessels) contained 20 L of test solution and were 

covered to reduce evaporation. Test vessels received no auxiliary aeration and were maintained 

at approximately 14°C in a temperature-controlled room.

Test Solution Preparation:

To prepare the 100 mg a.i./L test solution (which was corrected for a purity of 39.94%), a 

nominal amount of test substance (5007.5 mg) was dissolved in dilution water and the volume 

adjusted to 20 L. The solution was then stirred using a flat-bladed mixer for approximately 1 

minute to ensure homogeneity. 

Observations:

Any mortalities and/or sub-lethal effects were recorded at 3, 6, 24, 48, 72, and 96 hours after the 

start of exposure in the range-finding and definitive tests. The criteria for mortality included the 

absence of both respiratory movement and response to physical stimulation. 

Physico-Chemical Measurements:

Water temperature, pH, and dissolved oxygen concentrations were measured and recorded daily 

throughout the test. The temperature in the control replicate was also monitored hourly. 

Analysis of Test Solutions:

The stability and concentration of test substance in the 100 mg a.i./L solution was verified by 

chemical analysis (HPLC-MS) at 0 and 72 hours (fresh media) and 24 and 96 hours (old media). 

Duplicate samples, as well as samples taken at 24 (fresh media), 48 (old and fresh media), and 72 

(old media) hours, were stored frozen for further analysis if necessary.
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RESULTS

Endpoint value(s): Based on nominal concentrations, endpoint values from the definitive test 

were: 96-hour LC50 > 100 mg a.i./L; 96-hour NOEC = 100 mg a.i./L; 96-hour LOEC > 100 mg 

a.i./L

Results remarks: 

Range-Finding Test Results:

No mortalities or sub-lethal effects were observed during the range-finding test. Analytical 

measurements of the test substance in the 100 mg a.i./L test preparation at 0 and 24 hours ranged 

from 100 to 102% of nominal concentrations. Therefore, the test substance was considered to be 

stable under test conditions. 

Definitive Test Results: 

The test preparations were observed to be clear straw-colored solutions throughout the test. No 

mortalities occurred during the definitive test. One of the 10 fish in the 100 mg a.i./L group was 

observed to be sitting on the bottom of the tank at the 6-hour time point only. Given that this 

effect was not observed at any other time point during the study, it was considered to be due to 

natural causes and was therefore not considered to be an adverse effect.

Physico-Chemical Measurements:

Throughout the test, pH ranged from 7.5 to 7.8 and dissolved oxygen ranged from 8.4 to 10.5 

mg/L. At the start of the test, the temperature was 18°C; however, the study author indicated 

that this was because the logger had been moved from storage at a higher temperature and had 

not been able to acclimatize before this measurement was taken. Temperature of media was 

maintained at 13-14°C for the remainder of the test.

Verification of Test Concentrations: 

Analytical measurements of the test substance in fresh test preparations at the nominal 

concentration of 100 mg a.i./L were 77.6/89.4 mg a.i./L (duplicate samples) at 0 hours and 98.4 

mg a.i./L at 72 hours. Analytical measurements of the test substance in old (spent) test media at 

the nominal concentration of 100 mg a.i./L were 77.2 mg a.i./L at 24 hours and 94.5 mg a.i./L at 

96 hours. Regarding the value of 77.2 mg a.i./L observed in old test media at 24 hours, the study 

authors state: “Given that preliminary work conducted, along with the range finding test showed 

that the test item was stable in the test system, this result was considered to be anomalous and 

was considered not to have had an effect on the outcome of the study. Consequently, the results 

are based on nominal test concentrations only...” [Harris 2015a, p. 15]

CONCLUSIONS

In the definitive test, the 96-hour LC50 and LOEC for rainbow trout were > 100 mg a.i./L and the 

NOEC was 100 mg a.i./L, based on nominal concentrations.
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STUDY RELIABILITY

[2] Reliable with restrictions. The following deviations from test guidelines were noted: 

(1) OECD TG 203 recommends that the temperature of the test media for Oncorhynchus 

mykiss be within the range of 13-17°C during the test; however, a temperature of 18°C 

was measured at the start of the definitive test. The study author indicated that this was 

because the logger had been moved from storage at a higher temperature and had not 

been able to acclimatize before this measurement was taken. Temperature of the test 

media was maintained at 13-14°C for the remainder of the test. 

(2) OECD TG 203 states that if measured concentrations of the test substance (i.e., the active 

ingredient) deviate by more than 20% from the nominal concentrations throughout the 

test, results should be based on measured (rather than nominal) concentrations. In the 

definitive test, measured concentrations in one duplicate sample of the 0-hour (fresh)

sample and in the 24-hour (old) sample were lower than expected (77.6% and 77.2% of 

nominal concentrations, respectively). However, analysis of the duplicate 0-hour (fresh) 

sample showed a measured test substance concentration of 89.4% of the nominal

concentration. Analysis of the duplicate 24-hour sample was not possible as it was 

accidentally discarded.

1

 The low measured concentration in the 24-hour (old) sample 

might be anomalous given that (1) measured concentrations in the 72-hour (fresh) and 

96-hour (old) samples ranged from 94.5-98.4% of nominal and (2) measured 

concentrations in the range-finding test ranged from 100-102%, suggesting that the test 

substance is stable. 

REFERENCE

Harris, S. 2015a. Sodium glucoheptonate CAS No. 31138-65-5: Acute toxicity to rainbow trout. 

Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41402585. Sponsor: Harcros 

Chemicals, Inc., Dalton, GA. 

Text in quotes is taken directly from Harris (2015a).

 1 Although an explanation was not provided in the study report, Harcros Chemicals, Inc. notified EPA in an email 

dated April 20, 2015 (addressed from Ms. Ann Marie Savini of Harcros Chemicals, Inc. to Mr. John Schaeffer of 

EPA) that the duplicate 24-hour sample was discarded in error and could not be analyzed.
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D-2. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate 

Study 1: 48-Hour acute toxicity study in Daphnia magna (Harris, 2015b)

Title: Sodium glucoheptonate CAS No. 31138-65-5: Daphnia sp., 48-Hour acute 

immobilization test

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: D-gluco-heptonic acid, monosodium salt, (2.xi.)- (synonym: sodium glucoheptonate); 

CASRN 31138-65-5

Remarks: Amber-colored liquid; Batch No. T-461; Purity: 39.94%; 

Composition:

25.78% Beta Sodium Glucoheptonate (CASRN 30090-50-31)

14.16% Alpha Sodium Glucoheptonate (CASRN 13007-85-7)

10.96% Polysaccharides or Degradation Products

0.3 ppm Cyanide

1113 ppm Ammonia 

49.1% Water

pH = 9.1

Specific Gravity = 1.293

% Solids = 50.9%

No dextrose detected 

Based on the compositional information provided, the purity of the test substance was 39.94% 

a.i.2 (25.78% beta sodium glucoheptonate + 14.16% alpha sodium glucoheptonate).

METHOD

Method/guideline followed: OECD TG 202 (2004; “Daphnia sp., Acute Immobilisation Test”, 

Method C.2 of Commission Regulation (EC) No. 440/2008, and OPPTS 850.1010 (1996; 

“Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids”).

Test type: Acute Daphnid Toxicity Test (Static)

GLP compliant?: Yes

 1 30090-50-3 is not a valid CAS number according to the U.S. National Library of Medicine’s ChemIDplus website 

(http://chem.sis.nlm.nih.gov/chemidplus/); however, based on the chemical name provided (beta sodium 

glucoheptonate), this component is assumed to be a stereoisomer of CASRN 31138-65-5. 2 Active ingredient (a.i.) = CASRN 31138-65-5
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Year study was performed: 2015

Test species: Water flea (Daphnia magna); < 24 hours old; obtained from in-house laboratory 

cultures

Analytical monitoring: Yes

Exposure duration: 48 hours

Concentrations tested: Nominal: 0, 0.10, 1.0, 10, and 100 mg a.i./L (range-finding test); 

0 and 100 mg a.i./L (definitive test)

Test method/conditions remarks:

Dilution Water: 

Reconstituted water (ISO medium) was used for both the range-finding and definitive tests. The 

reconstituted water had an approximate theoretical total hardness of 250 mg CaCO3/L. The

reconstituted water was diluted 50:50 with deionized reverse osmosis water to achieve a total 

water hardness that complied with OPPTS-recommended water characteristics. At the start of 

the definitive test, the dilution water had a total water hardness of 146 mg CaCO3/L and an 

alkalinity of 27.6 mg CaCO3/L. 

Photoperiod:

For both the range-finding and definitive tests, the photoperiod was 16 hours light (intensity: not 

indicated for range-finding study; 565 to 626 lux for definitive study) and 8 hours darkness with 

20 minute dawn and dusk transition periods.

Range-Finding Test Procedure: 

In the range-finding test, groups of ten D. magna per vessel (one vessel per concentration) were 

exposed to the test substance at nominal concentrations of 0.10, 1.0, 10, and 100 mg a.i./L for 48 

hours under static conditions. A control group of ten D. magna was maintained under identical 

conditions but not exposed to the test substance. All test concentrations were corrected for a test 

substance purity of 39.94%.

Test Conditions:

Test vessels (250-mL glass jars) contained 200 mL of test media and were covered to reduce 

evaporation. Test vessels were maintained in a temperature-controlled room at 21°C.

Test Solution Preparation:

A nominal amount of test substance (250.4 mg) was dissolved in dilution water and the volume 

adjusted to 1 L to give the 100 mg a.i./L test concentration (corrected for a purity of 39.94%) 

from which a series of dilutions was made to obtain the remaining nominal concentrations of 

0.10, 1.0, and 10 mg a.i./L. Each prepared test solution was then inverted several times to ensure 

adequate mixing and homogeneity.
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Analysis of Test Solutions:

The stability and concentration of test substance in the 100 mg a.i./L solution was verified by 

chemical analysis (HPLC-MS) at 0 and 48 hours. “The analytical methodology was not 

validated prior to the analysis of range-finding samples; full validation was later conducted 

specifically at the test concentration employed in the definitive test.” [Harris 2015b, p. 32]

Definitive Test Procedure: 

Based on the results of the range-finding test, a limit test was conducted at a concentration of 100 

mg a.i./L. In the limit test, four replicates of five D. magna were exposed to the test substance at 

a nominal concentration of 100 mg a.i./L (corrected for a purity of 39.94%) for 48 hours under 

static conditions. Additionally, four control replicates of five D. magna were maintained under 

identical conditions but not exposed to the test substance. 

Test Conditions:

Test vessels (250-mL glass jars) contained 200 mL of test solution and were covered to reduce 

evaporation. Test vessels were not aerated and were maintained in a temperature-controlled 

room at 21°C.

Test Solution Preparation:

To prepare the 100 mg a.i./L test solution (corrected for a purity of 39.94%), a nominal amount 

of test substance (250.4 mg) was dissolved in dilution water and the volume adjusted to 1 L. The 

prepared test solution was then inverted several times to ensure adequate mixing and 

homogeneity.

Analysis of Test Solutions: 

Samples from the control and the 100 mg a.i./L test groups were taken for quantitative analysis 

(HPLC-MS) at 0 and 48 hours. Samples taken at 0 hours were from the bulk test preparation and 

samples taken at 48 hours were from the pooled replicates. The 48-hour samples were stored 

frozen prior to analysis; however, the 0-hour samples were not stored frozen for the initial 2 days 

after sampling due to a technical error. This was not considered to have had an impact on the 

outcome of the test given that the achieved measured concentrations were close to nominal

concentrations. Additionally, duplicate samples were taken and stored frozen for further analysis 

if necessary.

Observations:

Any immobilization or adverse effects were recorded at 24 and 48 hours in the range-finding and 

definitive tests. Daphnia were considered to be immobilized if they were unable to swim for 

approximately 15 seconds after gentle agitation. 

Physico-Chemical Measurements:

Water temperature was measured and recorded daily throughout the test and the temperature was 

also measured every hour in one control replicate. Dissolved oxygen concentrations and pH 

were measured and recorded at 0 and 48 hours. 
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RESULTS

Endpoint value(s): 

Based on nominal concentrations, endpoint values from the definitive test were:

48-hour EC50 > 100 mg a.i./L; 48-hour NOEC = 100 mg a.i./L; 48-hour LOEC > 100 mg a.i./L

Results remarks: 

Range-Finding Test Results:

The measured test substance concentration in the 100 mg a.i./L group was 93% of the nominal 

concentration at 0 hours and 87% of the nominal concentration at 48 hours. Therefore, the test 

substance was considered to be stable under test conditions. No immobilization was observed in 

the 0.10 or 1.0 mg a.i./L test groups. Immobilization was noted in a single daphnid each from 

the 10 and the 100 mg a.i./L test groups at 48 hours; however, this was not considered to be an 

adverse effect as no more than 10% immobilization occurred. Based on these findings, a single

limit concentration of 100 mg a.i./L was selected for the definitive test. 

Definitive Test Results: 

The test preparations were observed to be clear and colorless solutions throughout the test. No

immobilization or abnormal signs were observed during the definitive test. 

Physico-Chemical Measurements:

Throughout the test, temperature was maintained at 21°C, pH ranged from 7.5 to 8.3, and 

dissolved oxygen ranged from 8.4 to 9.1 mg/L. 

Verification of Test Concentrations: 

Analysis of the 100 mg a.i./L test solutions at 0 and 48 hours showed measured test 

concentrations to range from 80 to 86% of nominal concentrations; therefore, the results were 

based on nominal concentrations.

CONCLUSIONS

In the definitive test, the 48-hour EC50 and LOEC for Daphnia magna were > 100 mg a.i./L and 

the NOEC was 100 mg a.i./L, based on nominal concentrations.

STUDY RELIABILITY

[2] Reliable with restrictions. The following deviation from test guidelines was noted: 

According to Method C.2 of Commission Regulation (EC) No. 440/2008, the alkalinity of the 

dilution water should be 0.8 mmol/L; however, as noted by the study author, the alkalinity of the 

dilution water used in this test was 27.6 mg CaCO3/L (equivalent to 0.27576 mmol/L), which is 

less than the guideline-recommended value. 
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN

31138-65-5

13

REFERENCE

Harris, S. 2015b. Sodium glucoheptonate CAS No. 31138-65-5: Daphnia sp., 48-hour acute 

immobilization test. Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41402586. 

Sponsor: Harcros Chemicals, Inc., Dalton, GA. 

Text in quotes is taken directly from Harris (2015b).
Final HPV2 Test Rule Data Adequacy Review (August 2015) CASRN

31138-65-5

14

E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Aquatic 

Invertebrates

Yes Yes No

Toxicity to Aquatic Plantsa Yes Yes No

Health Effects

Acute Toxicitya Yes Yes No

Repeated-Dose Toxicitya Yes Yes No

Gene Mutationa Yes Yes No

Chromosomal Aberrationsa Yes Yes No

Reproductive Toxicitya Yes Yes No

Developmental Toxicitya Yes Yes No

a Data were reviewed in a separate report.