SRC TR

- 1 3

- 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 Sol utions Division

SRC, Inc.
7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027
Task FG007. 3 .009. 00 6

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460

Bryan Loba r , Project Officer
David Brooks , Work Assignment Manager Amy Benson , Alternate Work Assignment Manager

June 26

, 2013Final HPV3 Chemical Data Adequacy Review CASRN 98

- 09

- 9

i 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

................................ ................................ ........... 17Final HPV3 Chemical Data Adequacy Review CASRN 98

- 09

- 9

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission from Lanxess Corporation (dated March 26 , 20 13) that
contain s data from toxicity testing of b enzenesulfonyl 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 65 385 , October 21, 2 011; referred to herein as HPV3; available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

2009

- 0112

- 0086 as of June 25, 2013)

.

T he HPV 3 tes t rule requires testing with CASRN 98

- 09

- 9 for the following toxic ological endpoints: C2 testing requirement (consisting of acute toxicity to Daphnia and toxicity to algae testing if log K ow < 4.2 or chronic toxicity to Daphnia and toxicity to algae testin g if log K ow ≥ 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). A n experimentally measured log K ow value of 3.39 h as been reported for
CASRN 98

- 09

- 9 on the ECHA website 1 and the EPI Suite modeling program predicts a log K ow value of 2.94 2

. Both values indicate that the log K ow of CASRN 98

- 09

- 9 is < 4.2 ; therefore, the
C 2 aquatic toxicity testing requirement is consid ered to include acute toxicity to Daphnia and toxicity to algae testing

.

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

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 base d 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 requi rements. 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 in adequate, a basis for that conclusion is supplied. A table summarizing data availability/adequacy for the daphnid and algae SIDS endpoint s 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

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- 9

2 C

. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TES T RULE

Da ta supplied on March 26 , 2013 by Lanxess Corporation are adequate to satisfy the acute toxicity to D aphnia and toxicity to algae (C2) test ing requireme nt s promulgated in the final
HPV3 test rule

.

Data for the gene mutation ( E1 ) and chromosomal aberration (E2) test ing requirements were submitted by Lanxess on December 3, 2012 and December 17, 2012 , respectively (submissions
available online at http://www.regu lations.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, 2 013 ) 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 test ing 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

3 D . DATA FOR SIDS ENDPOINTS

D

- 1. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

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

Title: Benzenesulfonyl c hloride: Daphnia sp., 48

- hour acute immobilization test

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenesulfonyl c hlo ride

, 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 850.101 0, 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 ol d, bred at test facility

Analytical monitoring: Yes

. Analytical monitoring was conducted for the range

- finding and definitive test

s , 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 hydrochlor ic 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 Assessme nt 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

4 Concentrations tested: Range

- finding T est : Nominal concentrations of test material : 0 , 0.10, 1.0, 10, and 100 mg/L.
Mean m easured concent rations of hydrolysis product for nominal concentrations of 10 and 100 mg/L: 13.9 and 141.5 mg/L, respectively.
Initial E xperiment : Nominal concentrations of test material: 0, 10, 18, 32, 56, and 100 mg/L (analytical monitoring not conducted). Definitiv e Test : Nominal concentrations of test material : 0

, 32, 56, and 100 mg/L

. Mean

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

Test method/conditions remarks:
General Experimental Conditions : Reconstitute d 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 CaC O 3 therefore, in order to comply with the water characteristics for the OPPTS guideline, t he 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 Or ganic Carbon (TOC) concentration of 0.54 mg C/L.” [ V ryenhoef 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 approxima tely 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.” [ V ryenhoef 2013a

, p. 10]

After the daphnids were introduced in to 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

, w ith 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. ” [ V ryenhoef 2013a, p. 10]

In the initial experiment and defi nitive 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.” [ V ryenhoef 2013a

, p. 11]

Specific Experimental Conditions : Range

- finding Test : “In the range

- finding test

, Daphnia magna were exp osed 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 dilutio ns 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 ” [ V ryenhoef
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

5 “A sample o f 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.” [ V ryenhoef 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 resu lts of the range

- finding test .] At the start of the test 5 daphnids were place d 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 .” [ V ryen hoef 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 a djusted te st concentrations in order to determine the true toxic effect of the test item. ” 1
[ V ryenhoef 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 amo unt 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 t he test preparations was adjusted to pH 8.0 prior to the addition of the daphnids. ” [ V ryenhoef 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 2 5 daphnids/L]

. Four replicate test and control vessels were prepared .” [ V ryenhoef 2013a

, p. 12]

“ Water samples were taken from the control and each test group (replicates R 1

– R 4 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 2013 a , p. 13]

“ The concentration of benzenesulp honic 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 biolo gical part of this study was also used as the analytical standard. ” [ Vryenhoef 2013 a , p. 25] The limit of quantitation was 0.048 mg/L.

1 While the study author consider ed 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

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6 Evaluation of Data : Initial Experiment: An EC 50 value was not estimated in the study report. To calculate the EC 50 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) 1 at the 50% response level
for dichotomous models . The result of the model providing the best fit to the data (based upon evalu ation of p

- and AIC

- value s , and visual inspection ) was selected to estimate the EC 50 value. Definitive Test : “An estimate of the EC 50 values was given by inspection of the immobilization data.” [ V ryenhoef 2013a

, p. 13 ]

RESULTS

Endpo int value(s): Based on nominal concentrations: Initial Experiment (no pH adjustment to test water): 48

- hour EC 50 = 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 w ater adjusted to 8.0

- 8.1) : 48

- hour EC 50 > 100 mg/L , the highest
concentration tested ; 48

- hour NOEC = 100 mg/L

.

Results remarks: Range

- finding Test : The cumulative number s of immobiliz ed 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 degradatio n 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.” [ V ryenhoef 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 EC 50 of 46.7 mg/L based on nominal concentrations.

During t he initial experiment , the pH ranged from 2.8 to 7.8 ; pH decreased as test substance
concentration increased. T he 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

. T he dissolved oxygen concentration ranged from 8.5 to 9.0 mg O 2 /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

7 Defin itive 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 concen trations 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. ” [ V ryenhoef 2013a, p. 15]

“ Temper ature 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 Gui deline. 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 spec ification. ” [ V ryenhoef 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 O 2 /L.

“ Analysis of the 100 mg/L test preparation at 0 and 48 hours

... sho wed measured hydrolysis product concentrations of 109 mg/L and 103 mg/L respectively were obtained. ” [ V ryenhoef 2013a, p. 15]

CONCLUSIONS

For the initial e xperiment ( in which no pH adjustment was made to test water ), the 48

- hour EC 50 for Daphnia magna w as 46.7 mg/L ( based on nominal concentrations ; estimate d for this

summary using BMD software ). The 48

- hour NOEC and LOEC were 32 and 56 mg/L, respectively.

For the definitive t est ( in which the pH of the test water was adjusted to 8.0

- 8.1 prior to the st art of exposure ) , t he 48

- hour EC 50 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 subst ance 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 con centrations approximating target values.Final HPV3 Chemical Data Adequacy Review CASRN 98

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- 9

8 REFERENCE

Vryenhoef, H. ( 2013a ) Benezesulfonyl chloride: Daphnia sp., 48

- hour acute immobilization

test

. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study N o. 41201583

. Sponso r : Lanxess Corporation , Pittsburgh, PA

. Study report a vailable online at: http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0140 as of June
24 , 201 3.

Text in quotes taken directly from Vryenhoef ( 2013a )

.Final HPV3 Chemical Data Adequacy Review CASRN 98

- 09

- 9

9 D

- 2. Toxicity to Aquatic Plants

Summary of endpoint: Adequate

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

Title: Benz e n e s ulfonyl chloride: Algal inhibition t e st

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenesulfonyl c hloride

, CASRN 98

- 09

- 9

Remarks: Purity: 99.53%; Impurities: 0.043% benzenesulfonic acid, 0.04% diphenylsulfone,
0.03% chlorob enzene; 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 s tudy 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 experim entally 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 (200 0) , 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

10 Concentrations tested: Range

- finding T est : Nominal concentrations of test material : 0, 0.10, 1.0, 10, and 100 mg/L.
M ean m easured conce ntrations of hydrolysis pro duct for nominal concentrations of 10 and 100 mg/L: 13. 25 and 96.4 mg/L, respectively.
Initial E xperiment : Nominal concentrations of test material : 0, 6.25, 12.5, 25, 50

, and 100 mg/L (analytical monitoring not conducted)

. De finitive Test : Nominal concentrations of test material : 0, 6.25, 12.5, 25, 50

, and 100 mg/L.
M ean m easured concentration of hydrolysis pro duct for nominal concentration of 100 mg/L : 105 mg/L.

Test method/conditions remarks:
General Experimental Cond itions : 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 t o 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 identi cal conditions but not exposed to the test item. ” [ Vryenhoef 2013 b, 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 10 4 cells/mL. C ell density was “ d etermined 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 b y warm white lighting (380

- 730 nm) and constantly shaken at approximately 150 rpm for 96 hours. ” [ Vryenhoef
2013 b

, p. 11 ]

Cell density of each flask was determined at 0 and 96 hours for the rang e

- finding test and at 0, 24, 48, 72, and 96 hours for the in itial 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
2013 b

, p. 1 3 ]

Specific Experimental Conditions : Range

- finding Test : “An amount of test item (5 0 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 solution s was separately inoculated with 4.7 mL of algal suspension within 30 minutes of preparation

... ” [ Vryenhoef 2013 b, p

. 11 ]

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

, p

. 11 ]Final HPV3 Chemical Data Adequacy Review CASRN 98

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11 “A sample of each test concentr ation was taken for chemical analysis at 0 and 96 hours in order to determine the stability of the test item under test conditions. ” [ Vryenhoef 2013 b

, p

. 12 ]

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

vo lume 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 2013 b

, 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 10 5 cells per mL

. Inoculation of 500 mL of test medium w ith 18.7 mL of this algal suspension gave an initial nominal cell density of 1 x 10 4 cells per mL and [according to the study investigators] had no significant dilution effect on the final test concentration. ” [Vryenhoef 2013 b

, p. 12]

Definitive Tes t : “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 a djusted test concentra tions in order to determine the true toxic effect of the test item. ” 1 [Vryenhoef 2013 b

, p. 1 3 ]

“ For the purpose of the definitive test

... [a] n amount of test item (1 00 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 s olutions was separately inoculated with 10.0 mL of algal suspension within 30 minutes of preparation

... ” [ Vryenhoef 2013 b

, p

. 13 ] Test concentrations
for the definitive test were selected based on results of the initial experiment

.

“ Six flasks each contai ning 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for the treatment group. ” [Vryenhoef 2013 b

, p. 1 3 ]

“ Pre

- culture conditions gave an algal suspension in log phase growth characterized by a cell
de nsity of 5.00 x 10 5 cells 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 10 4 cells 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 consider ed 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

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12 “ Samples were taken from the control (replicates R 1

– R 6 pooled) and each test group (replicates
R 1

- R 3 pooled) at 0 and 96 hours for quantitative analy sis .” [ Vryenhoef 2013b

, p. 1 4 ]

V erification of t est c oncentrations : “ 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 t he 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 th is 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 concentra tions 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 Guideline s ).

 The mean of the coefficients of variation of the section by section specific g rowth 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 ex ceed 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 EC x 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 EC 50 values for any growth parameters . For this summary, the EPA reviewer calculated growt h 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 N n – ln N 1
t n – t 1

where: μ is the average specific growth rate from time t 1 to t n N 1 is the cell concentration at t 1 N n is the cell concentration at t n t 1 is the time of first measurement (0 hours)

t n is the time of n th measurement (96 hours)Final HPV3 Chemical Data Adequacy Review CASRN 98

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13 To calculate the EC 50 values for the initial experiment

, the EPA reviewer analyzed data for cell
densit y and growth rate using the most current version of EPA’s Benchmark Dose (BMD) software (US EPA 2012) 1 at the 50% response level for continuous variable models

. Adequacy of model fit was based on valuation of p

- and AIC

- values, and visual inspection.

D efi nitive T

est : “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 computeri s ed interpolation using the Xlfit software package (IDB S). ECx values were then determined from the equation for the fitted line. It was not possible to calculate 95% confidence
limit s for the EC 50 values as the data generated did not fit the models available for the calculation
of confidence limits

. ” [ Vrye nhoef 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]

R ESULTS

Endpoint value(s): B ased on nominal concentrations : Initial Experiment (no pH adjustment to test water) : 96

- hour EC 50 ( cell density): between 12.5 and 25 mg/L ; 96

- hour EC 50 (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 EC 50 (growth rate) > 100 mg/L; 96

- hour EC 50 (biomass) > 100 mg/L; 96

- hour EC 50 (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 m g/L.” [Vryenhoef 2013 b

, 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, re lative 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 con ditions were suitable for algal growth

.

Measured concentrations of hydrolysis pro duct 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

pro duct 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 te chnical 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

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14 respectively. Results indicate that the degradation product was stable over the 96

- hour test period.

Initial E xperiment : 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 Rate 1

(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 det ermine EC 50 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 EC 50 values for cell density a nd growth rate
could not be determined. Based on visual inspection, the 96

- hour EC 50 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 EC 50 value was also between 12.5 and 25 mg/L , with a NO EC 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 d efinitive t est (in which pH of test water was adjusted to 7.5 prior to the start of exposure),

t here 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 subcapi tata (CCAP 278/4) were not affected by the presence of the degradation product over the
96

- Hour exposure period.” [ Vryenhoef 2013 b, 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 2013 b, p. 21]

“ At the start of the test all control and test cultures were observed to be clear colorless solutions.
After the 96

- h our test period all control and test cultures were observed to be bright green dispersions .” [ Vryenhoef 2013 b, p. 21]Final HPV3 Chemical Data Adequacy Review CASRN 98

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15 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 concentration s 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.

 T he 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 i nitial e xperiment ( in which no pH adjustment was made to test water), the 96

- hour EC 50 value s for cell density and growth rate were between 12.5 and 25 mg/L , with NOEC s of 12.5 mg/L.

For the d efinitive t est ( in which pH of test water was adjusted to 7.5 prior to the start of

exposure )

, t he 96

- hour EC 50 values for growth r ate, 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 th e i nitial e xperiment ( with no pH adjustment to test water) showed that the test material produced a c oncentration

- 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 th e definitive test are not cons idered valid

. However,

r esults of the i nitial e xperime nt w hich was conducted following OECD TG 201 (i.e., no pH adjustment) are considered valid

. For the initial experiment

, EC 50 values for growth parameters were not reported , but d ata presented in the s tudy report were sufficient to determine a range for
the EC 50 value s for growth rate and cell density , although a single point estimate could not be
determined for either EC 50 value. Furthermore, a lthough 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 val ues. Therefore, this study is considered reliable with restrictions.Final HPV3 Chemical Data Adequacy Review CASRN 98

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16 REFERENCE

Vryenhoef, H. ( 2013 b ) Benzenesulfonyl chloride: Algal inhibition t est

. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Study N o. 41201584

. S ponsor: Lanxess Corporation

, Pittsburgh, PA

. Study report a vailable online at: http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2009

- 0112

- 0140 as of June
2 4 , 2013.

Text in quotes was taken directly from Vryenhoef ( 2013 b )

.Final HPV3 Chemical Data Adequacy Review CASRN 98

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17 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 Pla nts Yes Yes No