SRC TR-13-048

HPV3 TEST RULE DATA ADEQUACY REVIEW (FINAL):

TOXICITY TO DAPHNIA AND ALGAE

Benzenesulfonyl chloride, CASRN 98-09-9

Prepared by:

Ashlee Aldridge, Julie Klotzbach, William L. Richards, 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 FG007.3.009.006

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar, Project Officer

David Brooks, Work Assignment Manager

Amy Benson, Alternate Work Assignment Manager

June 26, 2013
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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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 Aquatic Invertebrates............................................................................ 3

D-2. Toxicity to Aquatic Plants................................................................................................. 9

E. DATA ADEQUACY SUMMARY TABLE........................................................................... 17
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission from Lanxess Corporation (dated March 26, 2013) that

contains data from toxicity testing of benzenesulfonyl chloride (CASRN 98-09-9) and was

received by EPA in response to the final test rule, “Testing of Certain High Production Volume 

Chemicals; Third Group of Chemicals” (76 FR 65385, October 21, 2011; referred to herein as 

HPV3; available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT2009-0112-0086 as of June 25, 2013). 

The HPV3 test rule requires testing with CASRN 98-09-9 for the following toxicological

endpoints: C2 testing requirement (consisting of acute toxicity to Daphnia and toxicity to algae 

testing if log Kow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testing if log Kow ≥ 

4.2), 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). An experimentally measured log Kow value of 3.39 has been reported for 

CASRN 98-09-9 on the ECHA website1and the EPI Suite modeling program predicts a log Kow

value of 2.942. Both values indicate that the log Kow of CASRN 98-09-9 is < 4.2; therefore, the 

C2 aquatic toxicity testing requirement is considered to include acute toxicity to Daphnia and 

toxicity to algae testing.

The March 26, 2013 submission from Lanxess Corporation (available online at http://www.

regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0140 as of June 25, 2013)

contains data from acute toxicity to Daphnia and toxicity to algae tests.

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

evaluated in this report. 

B. EVALUATING DATA ADEQUACY

Data available for the daphnid and algae SIDS toxicity 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 SIDS endpoints, study details were compared to 

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

Studies for a common endpoint are grouped together in the document and an endpoint adequacy 

assessment is provided near the top of the first page of the summaries for that endpoint. 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 the daphnid and algae SIDS 

endpoints is included on the last page of this document (Section E).

 

1 Available at http://apps.echa.europa.eu/registered/data/dossiers/DISS-9d8ca91a-d8a5-0cd4-e044-

00144f67d249/AGGR-e7cad612-601f-4077-bf75-88ef184348e6_DISS-9d8ca91a-d8a5-0cd4-e044-

00144f67d249.html#AGGR-e7cad612-601f-4077-bf75-88ef184348e6 as of June 25, 2013.

2 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 June 13, 2013.
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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

Data supplied on March 26, 2013 by Lanxess Corporation are adequate to satisfy the acute 

toxicity to Daphnia and toxicity to algae (C2) testing requirements promulgated in the final 

HPV3 test rule. 

Data for the gene mutation (E1) and chromosomal aberration (E2) testing requirements were 

submitted by Lanxess on December 3, 2012 and December 17, 2012, respectively (submissions 

available online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-

0112-0128 and http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-

0134, respectively, as of June 25, 2013) and were previously reviewed by EPA (review available 

online at http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0136 as 

of June 25, 2013) and determined to be adequate to satisfy the E1 and E2 testing requirements 

for CASRN 98-09-9.

Data have not yet been submitted for the F1 toxicity testing requirement, consisting of a 

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

test. In a letter to EPA dated October 23, 2012 (available online at http://www.regulations.gov/

#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0122 as of June 25, 2013), Lanxess 

Corporation indicated that the study report for F1 testing would be submitted to EPA by July 31, 

2013.
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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D. DATA FOR SIDS ENDPOINTS

D-1. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity to Daphnia magna (Vryenhoef, 2013a)

Title: Benzenesulfonyl chloride: Daphnia sp., 48-hour acute immobilization test

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenesulfonyl chloride, CASRN 98-09-9

Remarks: Purity: 99.53%; Impurities: 0.043% benzenesulfonic acid, 0.04% diphenylsulfone, 

0.03% chlorobenzene; Appearance: Clear, colorless liquid; Batch No.: KX120302.A.

METHOD

Method/guideline followed: OECD TG 202, OPPTS 850.1010, ASTM E729-96, Method C.2 

of Commission Regulation (EC) No. 440/2008

Test type: Daphnia sp., Acute Immobilization Test (Static)

GLP compliant?: Yes

Year study was performed: 2013

Test species: Water flea (Daphnia magna), approximately 24 hours old, bred at test facility

Analytical monitoring: Yes. Analytical monitoring was conducted for the range-finding and 

definitive tests, but not for the initial experiment. Due to the rapid hydrolysis of the test material 

(half-life < 5 min)1, analytical monitoring was conducted for the stable hydrolysis product 

benezenesulfonic acid.

2

Exposure duration: 48 hours

 

1 The experimentally measured hydrolysis half-life of benzenesulfonyl chloride at 25°C is 3.9 minutes. The 

hydrolysis products are benzenesulfonic acid and hydrochloric acid. (Haughton, AR; Laird, RM; Spence, MJ. 

(1975) Reactions of organic sulphur compounds. Part I. The hydrolysis of arenesulphonyl chlorides. J. Chem. 

Soc., Perkin Trans. 2, 6: 637-643)

2

In accordance with the OECD Series on Testing and Assessment Number 23: Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures (2000), it is appropriate to test the hydrolysis products of 

substances that have a half-life of less than 1 hour. A solution of degradation products may be obtained by leaving a 

stock or test solution for a period equal to 6 half-lives of the substance. 
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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Concentrations tested: 

Range-finding Test: Nominal concentrations of test material: 0, 0.10, 1.0, 10, and 100 mg/L. 

Mean measured concentrations of hydrolysis product for nominal concentrations of 10 and 100 

mg/L: 13.9 and 141.5 mg/L, respectively.

Initial Experiment: Nominal concentrations of test material: 0, 10, 18, 32, 56, and 100 mg/L 

(analytical monitoring not conducted). 

Definitive Test: Nominal concentrations of test material: 0, 32, 56, and 100 mg/L. Mean 

measured concentration of hydrolysis product for nominal concentration of 100 mg/L: 106 mg/L.

Test method/conditions remarks: 

General Experimental Conditions:

Reconstituted water was used for the range-finding test, initial experiment, and definitive test. 

“The theoretical total water hardness of reconstituted water is 250 mg/L as CaCO3 therefore, in 

order to comply with the water characteristics for the OPPTS guideline, the prepared 

reconstituted water was diluted in a ratio of 50:50 with deionized reverse osmosis water prior to 

use to reduce the total water hardness. At the start of the definitive test the diluent had a 

suspended solids content of 0.1 mg/L and a Total Organic Carbon (TOC) concentration of 0.54 

mg C/L.” [Vryenhoef 2013a, p. 10]

The test item was dissolved directly in reduced-hardness reconstituted water, using serial 

dilutions to yield final test concentrations. Glass jars (250 mL) containing approximately 200 

mL of test preparation were used as test chambers. “Each prepared concentration was inverted 

several times to ensure adequate mixing and homogeneity... The control group was maintained 

under identical conditions but not exposed to the test item.” [Vryenhoef 2013a, p. 10]

After the daphnids were introduced into the test solutions, the test vessels were covered to reduce 

evaporation and were maintained in a temperature-controlled room at ~20-22°C with a 

photoperiod of 16 hours light: 8 hours dark, with 20-minute dawn and dusk transition periods, 

for a total of 48 hours. “The daphnids were not individually identified, received no food during 

exposure and the test vessels were not aerated.” [Vryenhoef 2013a, p. 10]

In the initial experiment and definitive test, “[w]ater temperature and light intensity were 

recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at 

the start and termination of the test. The pH and dissolved oxygen concentration were measured 

using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna 

Instruments HI 93510 digital thermometer.” [Vryenhoef 2013a, p. 11]

Specific Experimental Conditions:

Range-finding Test: “In the range-finding test, Daphnia magna were exposed to a series of 

nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L... An amount of the test item (50 mg) 

was dissolved in reconstituted water and the volume adjusted to 500 mL to give the 100 mg/L 

test concentration from which a series of dilutions was made to give further test concentrations 

of 10, 1.0, and 0.10 mg/L... 10 daphnids were placed in each test and control vessel” [Vryenhoef 

2013a, p. 10] The loading was 50 daphnids/L. Replicates were not used in the range-finding 

test. 
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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“A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order 

to determine the stability of the test item under test conditions.” [Vryenhoef 2013a, p. 10]

Initial Experiment: “An amount of test item (250 mg) was dissolved directly in reconstituted 

water and the volume adjusted to 2.5 liters to give the 100 mg/L test concentration from which a 

series of dilutions was made to give further test concentrations of 56, 32, 18 and 10 mg/L...

[These concentrations were selected based on results of the range-finding test.] At the start of 

the test 5 daphnids were placed in each test and control vessel at random, in the test preparations

[resulting in a loading of 25 daphnids/L]. Four replicate test and control vessels were prepared.” 

[Vryenhoef 2013a, p. 11]

Definitive Test: “A [test substance] concentration dependent decline in pH was observed at 0 

hours during the initial experiment in the range of pH 7.2 - 7.4 at 10 mg/L through to pH 2.8 -

2.9 at 100 mg/L. It was therefore considered that the immobilization observed at the test 

concentrations of 56 and 100 mg/L may have been due to the acidic nature of the test item rather 

than its true toxicity. As such it was considered appropriate to perform the definitive test using 

pH adjusted test concentrations in order to determine the true toxic effect of the test item.”

1

[Vryenhoef 2013a, p. 12] Concentrations for the definitive test were selected based on results of 

the initial experiment.

“For the purpose of the definitive test... [a]n amount of test item (200 mg) was dissolved in 

reconstituted water and the volume adjusted to 2 liters to give the 100 mg/L test concentration 

from which a series of dilutions was made to give further test concentrations of 56 and 32 mg/L. 

The pH of each of the test preparations was adjusted to pH 8.0 prior to the addition of the 

daphnids.” [Vryenhoef 2013a, p. 12]

“At the start of the test 5 daphnids were placed in each test and control vessel at random, in the 

test preparations [resulting in a loading of 25 daphnids/L]. Four replicate test and control vessels 

were prepared.” [Vryenhoef 2013a, p. 12]

“Water samples were taken from the control and each test group (replicates R1– R4 pooled) at 0 

and 48 hours for quantitative analysis.” [Vryenhoef 2013a, p. 13] 

Verification of Test Concentrations: “All samples were stored at approximately -20 °C prior to 

analysis. Samples at the No Observed Effect Concentration and above only were analyzed.”

[Vryenhoef 2013a, p. 13] 

“The concentration of benzenesulphonic acid in the test samples was determined by high 

performance liquid chromatography with UV detection (HPLC/UV) using an external standard. 

The test item gave a chromatographic profile consisting of a single peak... The test item 

described in the biological part of this study was also used as the analytical standard.” 

[Vryenhoef 2013a, p. 25] The limit of quantitation was 0.048 mg/L.

 

1 While the study author considered the pH effect to not be a “true” toxic effect, EPA disagrees. Any toxicity 

resulting from a change in pH caused by the test substance is considered by EPA to be a true toxic effect.
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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Evaluation of Data: 

Initial Experiment: An EC50 value was not estimated in the study report. To calculate the EC50

value for this summary, the EPA reviewer analyzed immobility data using the most current 

version of EPA’s Benchmark Dose (BMD) software (US EPA 2012)1at the 50% response level

for dichotomous models. The result of the model providing the best fit to the data (based upon 

evaluation of p- and AIC-values, and visual inspection) was selected to estimate the EC50 value.

Definitive Test: “An estimate of the EC50 values was given by inspection of the immobilization 

data.” [Vryenhoef 2013a, p. 13]

RESULTS

Endpoint value(s): Based on nominal concentrations:

Initial Experiment (no pH adjustment to test water): 48-hour EC50 = 46.7 mg/L (calculated for 

this summary using BMD software); 48-hour NOEC = 32 mg/L; 48-hour LOEC = 56 mg/L. 

Definitive Test (pH of test water adjusted to 8.0-8.1): 48-hour EC50 > 100 mg/L, the highest 

concentration tested; 48-hour NOEC = 100 mg/L. 

Results remarks: 

Range-finding Test:

The cumulative numbers of immobilized daphnids in the range-finding test at nominal 

concentrations of 0 (control), 0.10, 1.0, 10, and 100 mg/L were 0, 0, 0, 0, and 10 out of 10 initial 

daphnids at 48 hours. All daphnids at 100 mg/L had died after 24 hours. 

“Chemical analysis of the 10 and 100 mg/L test preparations at 0 hours showed measured 

degradation product concentrations of 14 and 138 mg/L, respectively, were obtained whilst 

concentrations of 14 [rounded off from 13.8] and 145 mg/L respectively were obtained at 48 

hours. These results indicated that the degradation product was stable over the test period.”

[Vryenhoef 2013a, p. 14]

Initial Experiment: 

The incidence of immobilization in the initial experiment at nominal concentrations of 0 

(control), 10, 18, 32, 56, and 100 mg/L, respectively, was 0, 0, 0, 0, 85, and 100% after 24 hours 

and 0, 0, 0, 0, 100, and 100% after 48 hours. Benchmark dose analysis of immobility data 

identified the gamma model as the best fit to the data (p-value = 0.8548; AIC = 22.2518), with an 

estimated 48-hour EC50 of 46.7 mg/L based on nominal concentrations.

During the initial experiment, the pH ranged from 2.8 to 7.8; pH decreased as test substance 

concentration increased. The pH (mean of four replicates) at nominal concentrations of 0, 10, 

18, 32, 56, and 100 mg/L, respectively, was 7.8, 7.3, 6.9, 6.0, 3.5, and 2.8 at 0 hours and 7.6, 7.6, 

7.6, 7.4, 3.6, and 3.0 after 48 hours. The dissolved oxygen concentration ranged from 8.5 to 9.0 

mg O2/L and the test temperature ranged from 20 to 22°C. 

 

1 U.S. EPA. (2012) Benchmark dose technical guidance document, version 2.2.0. Washington, DC: U.S. 

Environmental Protection Agency, Risk Assessment Forum. EPA/100/R-12/001. 
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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Definitive Test: 

The incidence of immobilization in the definitive test (in which the pH of the test water was 

adjusted to 8.0-8.1 prior to the start of exposure) at nominal concentrations of 0, 32, 56, and 100 

mg/L was 0%, 5%, 5%, and 5%, respectively, after 48 hours. The immobilization of 1/20 

daphnids at three test concentrations was considered by the study authors to be incidental and 

unrelated to exposure to the test substance.

“The test preparations were observed to be clear colorless solutions throughout the duration of 

the test.” [Vryenhoef 2013a, p. 15]

“Temperature was maintained at 20 °C to 21 °C throughout the test, while there were no 

treatment related differences for oxygen concentration or pH. Due to a technical oversight the 

temperature in a single vessel was not logged hourly as required by the Test Guideline. This was 

considered to have had no adverse effect on the outcome of the test given that temperature 

measurements taken from each of the individual test vessels over the 48-Hour test period showed 

that temperature remained within the guideline specification.” [Vryenhoef 2013a, p. 15]

Over the course of the 48-hour exposure period, the pH ranged from 7.7 to 8.1 and the dissolved 

oxygen concentration ranged from 8.6 to 9.2 mg O2/L. 

“Analysis of the 100 mg/L test preparation at 0 and 48 hours... showed measured hydrolysis 

product concentrations of 109 mg/L and 103 mg/L respectively were obtained.” [Vryenhoef

2013a, p. 15]

CONCLUSIONS

For the initial experiment (in which no pH adjustment was made to test water), the 48-hour EC50

for Daphnia magna was 46.7 mg/L (based on nominal concentrations; estimated for this 

summary using BMD software). The 48-hour NOEC and LOEC were 32 and 56 mg/L, 

respectively.

For the definitive test (in which the pH of the test water was adjusted to 8.0-8.1 prior to the start 

of exposure), the 48-hour EC50 for Daphnia magna was greater than 100 mg/L (based on 

nominal concentrations). The 48-hour NOEC was 100 mg/L, the highest concentration tested.

STUDY RELIABILITY

[2] Reliable with restrictions. Although the test substance concentration was not verified 

analytically for the initial experiment, measurements of the hydrolysis product for the rangefinding and definitive tests suggest that the methods of test solution preparation were adequate to 

achieve test substance concentrations approximating target values.
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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REFERENCE

Vryenhoef, H. (2013a) Benezesulfonyl chloride: Daphnia sp., 48-hour acute immobilization 

test. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 

41201583. Sponsor: Lanxess Corporation, Pittsburgh, PA. Study report available online at: 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0140 as of June 

24, 2013. 

Text in quotes taken directly from Vryenhoef (2013a).
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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D-2. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

Study 1: Toxicity to Pseudokirchneriella subcapitata (Vryenhoef, 2013b)

Title: Benzenesulfonyl chloride: Algal inhibition test

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenesulfonyl chloride, CASRN 98-09-9

Remarks: Purity: 99.53%; Impurities: 0.043% benzenesulfonic acid, 0.04% diphenylsulfone, 

0.03% chlorobenzene; Appearance: Clear, colorless liquid; Batch No.: KX120302.A

METHOD

Method/guideline followed: OECD TG 201, OPPTS 850.5400, ASTM E1218-04

Test type: Freshwater Alga and Cyanobacteria, Growth Inhibition Test (Static)

GLP compliant?: Yes

Year study was performed: 2013

Test species: Freshwater green algae (Pseudokirchneriella subcapitata), strain CCAP 278/4,

liquid cultures obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS 

Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. 

Analytical monitoring: Yes. Analytical monitoring was conducted for the range-finding and 

definitive tests, but not for the initial experiment. Due to the rapid hydrolysis of the test material 

(half-life < 5 min)1, analytical monitoring was conducted for the stable hydrolysis product 

benezenesulfonic acid.

2

Exposure duration: 96 hours

 

1 The experimentally measured hydrolysis half-life of benzenesulfonyl chloride at 25°C is 3.9 minutes. The 

hydrolysis products are benzenesulfonic acid and hydrochloric acid. (Haughton, AR; Laird, RM; Spence, MJ. 

(1975) Reactions of organic sulphur compounds. Part I. The hydrolysis of arenesulphonyl chlorides. J. Chem. 

Soc., Perkin Trans. 2, 6: 637-643)

2

In accordance with the OECD Series on Testing and Assessment Number 23: Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures (2000), it is appropriate to test the hydrolysis products of 

substances that have a half-life of less than 1 hour. A solution of degradation products may be obtained by leaving a 

stock or test solution for a period equal to 6 half-lives of the substance. 
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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Concentrations tested: 

Range-finding Test: Nominal concentrations of test material: 0, 0.10, 1.0, 10, and 100 mg/L. 

Mean measured concentrations of hydrolysis product for nominal concentrations of 10 and 100 

mg/L: 13.25 and 96.4 mg/L, respectively. 

Initial Experiment: Nominal concentrations of test material: 0, 6.25, 12.5, 25, 50, and 100 mg/L

(analytical monitoring not conducted). 

Definitive Test: Nominal concentrations of test material: 0, 6.25, 12.5, 25, 50, and 100 mg/L. 

Mean measured concentration of hydrolysis product for nominal concentration of 100 mg/L: 105

mg/L. 

Test method/conditions remarks: 

General Experimental Conditions:

Culture medium was prepared according to ASTM E1218-04 with deionized water purified using 

reverse osmosis. Tests were “conducted in 250 mL glass conical flasks each containing 100 mL 

of test preparation and plugged with polyurethane foam bungs to reduce evaporation... The test 

item was dissolved directly in culture medium... The stock solutions of each of the prepared 

concentrations were inverted several times to ensure adequate mixing and homogeneity. The 

control group was maintained under identical conditions but not exposed to the test item.”

[Vryenhoef 2013b, p. 11]

At the initiation of each exposure, the test medium was inoculated with an algal suspension to 

give an initial cell density of approximately 1 x 104cells/mL. Cell density was “determined 

using a Coulter Multisizer Particle counter. The flasks were then plugged with polyurethane 

foam bungs and incubated (INFORS Multitron Version 2 Incubator) at 24  2C under 

continuous illumination (intensity approximately 4300 lux) provided by warm white lighting 

(380-730 nm) and constantly shaken at approximately 150 rpm for 96 hours.” [Vryenhoef 

2013b, p. 11]

Cell density of each flask was determined at 0 and 96 hours for the range-finding test and at 0, 

24, 48, 72, and 96 hours for the initial experiment and definitive test. 

In the initial experiment and definitive test, “[t]he pH of the control and each test preparation was 

determined at initiation of the test and after 96 hours exposure. The pH was measured using a 

WTW pH 320 pH meter. The temperature within the incubator was recorded hourly using the 

incubators’ internal pt-100 temperature probe and Multicom Software Package.” [Vryenhoef 

2013b, p. 13]

Specific Experimental Conditions:

Range-finding Test: “An 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 4.7 mL of algal suspension within 30 

minutes of preparation...” [Vryenhoef 2013b, p. 11]

“Two replicate flasks were used for each control and test concentration.” [Vryenhoef 2013b, p. 

11] 
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

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“A sample of each test concentration was taken for chemical analysis at 0 and 96 hours in order 

to determine the stability of the test item under test conditions.” [Vryenhoef 2013b, p. 12]

Initial Experiment: “An amount of test item (100 mg) was dissolved in culture medium and the 

volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions 

were made to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. An aliquot (500 mL) of 

each of the stock solutions was separately inoculated with 18.7 mL of algal suspension within 30 

minutes of preparation...” [Vryenhoef 2013b, p. 12] Test concentrations for the initial 

experiment were selected based on results of the range-finding test.

“Six flasks each containing 100 mL of test preparation were used for the control and three flasks 

each containing 100 mL were used for the treatment group.” [Vryenhoef 2013b, p. 12]

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

density of 2.68 x 105cells per mL. Inoculation of 500 mL of test medium with 18.7 mL of this 

algal suspension gave an initial nominal cell density of 1 x 104cells per mL and [according to the 

study investigators] had no significant dilution effect on the final test concentration.”

[Vryenhoef 2013b, p. 12]

Definitive Test: “A [test substance] concentration dependent decline in pH was observed at 0 

hours during the initial experiment in the range of pH 7.4 at 6.25 mg/L through to pH 2.5 at 100 

mg/L. It was therefore considered that the inhibition of growth observed at the test

concentrations of 25, 50 and 100 mg/L may have been due to the acidic nature of the test item 

rather than its true toxicity. As such it was considered appropriate to perform the definitive test

using pH adjusted test concentrations in order to determine the true toxic effect of the test item.”

1

[Vryenhoef 2013b, p. 13]

“For the purpose of the definitive test... [a]n amount of test item (100 mg) was dissolved in 

culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which 

a series of dilutions was made to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. The 

pH of each of the stock solutions was measured and adjusted to pH 7.5. An aliquot (500 mL) of 

each of the pH adjusted stock solutions was separately inoculated with 10.0 mL of algal 

suspension within 30 minutes of preparation...” [Vryenhoef 2013b, p. 13] Test concentrations 

for the definitive test were selected based on results of the initial experiment.

“Six flasks each containing 100 mL of test preparation were used for the control and three flasks 

each containing 100 mL were used for the treatment group.” [Vryenhoef 2013b, p. 13]

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

density of 5.00 x 105cells per mL. Inoculation of 500 mL of test medium with 10.0 mL of this 

algal suspension gave an initial nominal cell density of 1 x 104cells per mL and [according to the 

study investigators] had no significant dilution effect on the final test concentration.” 

[Vryenhoef 2013b, p. 13]

 

1 While the study author considered the pH effect to not be a “true” toxic effect, EPA disagrees. Any toxicity 

resulting from a change in pH caused by the test substance is considered by EPA to be a true toxic effect.
Final HPV3 Chemical Data Adequacy Review CASRN 98-09-9

12

“Samples were taken from the control (replicates R1 – R6 pooled) and each test group (replicates 

R1 - R3 pooled) at 0 and 96 hours for quantitative analysis.” [Vryenhoef 2013b, p. 14]

Verification of test concentrations: “All samples were stored at approximately -20 °C prior to 

analysis... Only samples at the No Observed Effect Concentration (NOEC) and above were 

analyzed.” [Vryenhoef 2013b, p. 14] 

“The concentration of benzenesulphonic acid in the test samples was determined by high 

performance liquid chromatography with UV detection (HPLC/UV) using an external standard. 

The test item gave a chromatographic profile consisting of a single peak... The test item 

described in the biological part of this study was also used as the analytical standard.” 

[Vryenhoef 2013b, p. 36] The limit of quantitation was 0.048 mg/L.

Validation Criteria: “The results of the test are considered valid if the following performance 

criteria were met:

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

after 72 hours (OECD/EC Guidelines) and by a factor of at least 100 after 96 hours 

(EPA/OPPTS Guidelines).

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

the control cultures during the course of the test (0-24, 24-48 and 48-72 hours) must not 

exceed 35% (OECD Guidelines).

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

cultures after 72 hours must not exceed 7% (OECD Guidelines).

 The coefficient of variation of the cell density values in replicate control cultures must 

not exceed 20% after 72 and 96 hours (EPA/OPPTS Guidelines).” [Vryenhoef 2013b, p. 

17]

Determination of ECx values:

Initial Experiment: The study report included a detailed data table for cell density, but did not 

include calculations of growth parameters (i.e., growth rate, biomass, and yield) or EC50 values 

for any growth parameters. For this summary, the EPA reviewer calculated growth rate (from 0 

to 96 hours) for the initial experiment using the reported cell density data and the following 

equation (from Vryenhoef 2013b):

μ = ln Nn – ln N1

 tn – t1

where:

μ is the average specific growth rate from time t1 to tn

N1 is the cell concentration at t1

Nn is the cell concentration at tn

t1 is the time of first measurement (0 hours)

tn is the time of nth measurement (96 hours)
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To calculate the EC50 values for the initial experiment, the EPA reviewer analyzed data for cell 

density and growth rate using the most current version of EPA’s Benchmark Dose (BMD) 

software (US EPA 2012)1at the 50% response level for continuous variable models. Adequacy

of model fit was based on valuation of p- and AIC-values, and visual inspection.

Definitive Test: “For each individual test vessel (mean values for yield), percentage inhibition 

(arithmetic axis) was plotted against test concentrations (logarithmic axis) and a line fitted by 

computerised interpolation using the Xlfit software package (IDBS). ECx values were then 

determined from the equation for the fitted line. It was not possible to calculate 95% confidence 

limits for the EC50 values as the data generated did not fit the models available for the calculation 

of confidence limits.” [Vryenhoef 2013b, p. 16]

Statistical analysis: “One way analysis of variance incorporating Bartlett's test for homogeneity 

of variance... and Dunnett's multiple comparison procedure for comparing several treatments 

with a control... was carried out on the growth rate, yield and biomass integral data [from the 

definitive test] after 72 and 96 hours for the control and all test concentrations to determine any 

statistically significant differences between the test and control groups.” [Vryenhoef 2013b, pp. 

16-17]

RESULTS

Endpoint value(s): Based on nominal concentrations:

Initial Experiment (no pH adjustment to test water): 96-hour EC50 (cell density): between 12.5 

and 25 mg/L; 96-hour EC50 (growth rate): between 12.5 and 25 mg/L; 96-hour NOEC (cell 

density) = 12.5 mg/L; 96-hour NOEC (growth rate) = 12.5 mg/L.

Definitive Test (pH of test water adjusted to 7.5): 96-hour EC50 (growth rate) > 100 mg/L; 96-

hour EC50 (biomass) > 100 mg/L; 96-hour EC50 (yield) > 100 mg/L; 96-hour NOEC (growth 

rate) = 100 mg/L; 96-hour NOEC (biomass) = 100 mg/L; 96-hour NOEC (yield) = 100 mg/L.

Results remarks: 

Range-finding test: 

“The results showed no effect on growth 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 2013b, p. 18]

After 96 hours, mean inhibition of growth rate, relative to controls, was 2, 2, 0, and 84% at 

nominal test substance concentrations of 0.1, 1.0, 10, and 100 mg/L, respectively. After 96 

hours, mean inhibition of biomass, relative to controls, was 5, 3, 4, and 99% at nominal test 

substance concentrations of 0.1, 1.0, 10, and 100 mg/L, respectively.

The cell density of the control cultures increased by a factor of 251x over the 96-hour test period, 

indicating that culture conditions were suitable for algal growth. 

Measured concentrations of hydrolysis product at 0 and 96 hours for the 10 mg/L nominal 

concentration were 13.1 and 13.4 mg/L, respectively. Measured concentrations of hydrolysis 

product at 0 and 96 hours for the 100 mg/L nominal concentration were 95.4 and 97.4 mg/L, 

 

1 U.S. EPA. (2012) Benchmark dose technical guidance document, version 2.2.0. Washington, DC: U.S. 

Environmental Protection Agency, Risk Assessment Forum. EPA/100/R-12/001. 
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respectively. Results indicate that the degradation product was stable over the 96-hour test 

period.

Initial Experiment: 

The means ± standard deviations (SD) for cell density and growth rate, as well as percent 

inhibition of cell density and growth rate, as calculated by the EPA reviewer, are shown in 

Table 1. 

Table 1. Summary of Data for Cell Density and Growth in the Initial Experiment

Nominal 

Concentration 

(mg/L)

pH Cell Density 

(cells/mL) at 

96 Hours

(mean ± SD)

% Inhibition 

of Cell 

Density

Growth Rate1

(cells/mL/hr)

(mean ± SD)

% Inhibition 

of Growth 

Rate

0 Hours 96 Hours

Control 7.6 8.0 2320000 ± 90900 - 0.057 ± 0.001 -

6.25 7.4 8.1 2190000 ± 70000 5 0.056 ± 0.001 2

12.5 7.2 8.1 2250000 ± 107000 3 0.056 ± 0.001 2

25 3.7 3.8 25000 ± 24800 99 0.006 ± 0.010 90

50 3.0 3.0 4960 ± 2560 100 -0.008 ± 0.007 113

100 2.5 2.6 3270 ± 1090 100 -0.011 ± 0.003 119

1 Growth rate from 0 to 96 hours was calculated by the EPA reviewer.

To determine EC50 values for this review, BMD modeling (US EPA, 2012) was conducted for 

cell density and growth rate. However, none of the continuous variable models provided 

adequate fit to the data; therefore, point estimates of EC50 values for cell density and growth rate 

could not be determined. Based on visual inspection, the 96-hour EC50 value for cell density was 

between 12.5 and 25 mg/L, with a NOEC value of 12.5 mg/L. For growth rate, the 96-hour EC50

value was also between 12.5 and 25 mg/L, with a NOEC value of 12.5 mg/L.

The cell density of the control cultures increased by a factor of 234x over the 96-hour test period, 

indicating that culture conditions were suitable for algal growth. 

Definitive Test: 

In the definitive test (in which pH of test water was adjusted to 7.5 prior to the start of exposure),

there were no statistically significant decreases (p ≥ 0.05) in biomass, yield, or growth rate 

between the control and all test groups at 72 or 96 hours.

Data show “that the growth rate (r), yield (y) and biomass integral (b) of Pseudokirchneriella 

subcapitata (CCAP 278/4) were not affected by the presence of the degradation product over the 

96-Hour exposure period.” [Vryenhoef 2013b, p. 20]

“All test and control cultures were inspected microscopically at 96 hours. There were no 

abnormalities detected in any of the control or test cultures.” [Vryenhoef 2013b, p. 21]

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

After the 96-hour test period all control and test cultures were observed to be bright green 

dispersions.” [Vryenhoef 2013b, p. 21]
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Temperature was maintained between 22.5 and 23.5 oC throughout the definitive test. The pH 

was 7.5 in all cultures at time 0 and was 7.7-7.8 at 96 hours.

Measured concentrations of hydrolysis product at 0 and 96 hours for the 100 mg/L nominal 

concentration were 104 and 106 mg/L, respectively. Results indicate that the degradation 

product was stable over the 96-hour test period.

Validation Criteria: The cell growth observed in the control cultures during the definitive test

satisfied the criteria for a valid test.

 The cell concentration increased by a factor of 80 during the first 72 hours of the test and 

by a factor of 272 after 96 hours. 

 The mean coefficient of variation for section-by-section specific growth rate was 16% 

from 0 to 72 hours.

 The coefficient of variation for average specific growth rate was 3% from 0 to 72 hours.

 The coefficient of variation of the cell density was 16% after 72 hours and 5% after 96 

hours. 

CONCLUSIONS

For the initial experiment (in which no pH adjustment was made to test water), the 96-hour EC50

values for cell density and growth rate were between 12.5 and 25 mg/L, with NOECs of 12.5

mg/L. 

For the definitive test (in which pH of test water was adjusted to 7.5 prior to the start of 

exposure), the 96-hour EC50 values for growth rate, yield, and biomass were greater than 100 

mg/L. The 96-hour NOECs for growth rate, yield, and biomass were 100 mg/L. 

STUDY RELIABILITY 

[2] Reliable with restrictions. Results of the initial experiment (with no pH adjustment to test 

water) showed that the test material produced a concentration-related decrease in pH. Due to this 

observation, study authors adjusted pH of the test water in the definitive test to 7.5. However, 

OECD TG 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) does not include a 

procedure for adjustment of pH due to a pH-lowering effect of the test material. Due to this 

deviation from the test guideline, results of the definitive test are not considered valid. However, 

results of the initial experiment which was conducted following OECD TG 201 (i.e., no pH 

adjustment) are considered valid. For the initial experiment, EC50 values for growth parameters

were not reported, but data presented in the study report were sufficient to determine a range for 

the EC50 values for growth rate and cell density, although a single point estimate could not be 

determined for either EC50 value. Furthermore, although the test substance concentration was 

not verified analytically for the initial experiment, measurements of the hydrolysis product for 

the range-finding and definitive tests suggest that the methods of test solution preparation were 

adequate to achieve test substance concentrations approximating target values. Therefore, this 

study is considered reliable with restrictions.
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REFERENCE

Vryenhoef, H. (2013b) Benzenesulfonyl chloride: Algal inhibition test. Performing Laboratory: 

Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study No. 41201584. Sponsor: Lanxess 

Corporation, Pittsburgh, PA. Study report available online at: 

http://www.regulations.gov/#!documentDetail;D=EPA-HQ-OPPT-2009-0112-0140 as of June 

24, 2013. 

Text in quotes was taken directly from Vryenhoef (2013b).
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E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Aquatic Invertebrates Yes Yes No

Toxicity to Aquatic Plants Yes Yes No