# Endpoint study record: Toxicity to aquatic algae an d

cyanobacteria Algal Inhibition Test Benzenesulfonyl chloride Lanxess Corporation 41201584

Administrative Data

Data source

Reference

Data access

Data protection claimed



# Materials and methods

Test guideline

GLP compliance



## Test materials
Test material identity

Test material form

Details on test material UUID IUC5

- 7e15c24b

- 2ffa

- 417f

- 9cc4

- 75cdbf9436c3

Dossier UUID 0

Author admin4 / Harlan Laboratories Ltd. / Other / other:

Date 2013

- 03

- 25 12:36:57 GMT

Remarks

Purpose flag robust study summary
Study result type experimental result Study

period Experimental Starting Date: 13 September 2012 Exper imental

Completion Date: 19 February 2013

Reliability 1 (reliable without restriction)

Rationale for reliability incl. deficiencies Study conducted in compliance with agreed protocols , with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of th e relevant results.

Reference type Author Year Title Bibliographic

source Testing

laboratory Report

no. Owner

company Company

study no. Report

date

study report Vryenhoef

H 2013 Algal Inhibition

Test Benzenesulfonyl chloride Harlan Laboratories Ltd. Shardlow Business Park Shardlow Derbyshire DE72 2GD UK 41201584 Lanxess Corporation 111 RIDC Park West Drive Pittsburgh PA 15275-1112 UNITED STATES OF AMERICA

data submitter is data owner

yes, but willing to share

Qualifier Guideline Deviations

according to OECD Guideline 201 (Alga, Growth Inhibition Test) no

according to EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) no

according to other guideline: ASTME1218-04 no

yes (incl. certificate)

Identifier Identity

Common name Benzenesulfonyl chloride

other: clear colorless liquid

The integrity of the supplied data relating to the identity, purity and stability of the test item is the responsibility of the Sponsor. A Certificate of Analysis for the test item, provided by the Sponsor is given in the attached Appendix 1.

Identification: Benzenesulfonyl chloride Description: clear colorless liquid Batch: KX120302.A Purity: 99.53% Expiry / retest date: 12 April 2013 

# Page 1 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

### Confidential details on test material

Analytical monitoring

Details on sampling

Details on analytical methods Storage conditions: room temperature in the dark

Not applicable

yes

Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R 3 pooled) at 0 and 96 hours for quantitative analysis. All samples were stored at a pproximately -20 °C prior to analysis. Duplicate sa mples were taken at 0 and 96 hours and stored at approximately -20 oC for further analysis if necessary. Only samples at the No Observed Effect Concentration (NOEC) and above w ere analyzed.

The method of analysis, recovery and test preparati on analyses are described in the attached Appendix 4.

The test item is known to hydrolyse quickly in the presence of water to the corresponding benzenesulph onic acid (half-life of approximately 5 minutes). It is understood that the peak observed in the chromatograms was not the original benzenes ulphonyl chloride but was actually benzenesulphonic acid, which is the stable degradat ion product.

The concentration of benzenesulphonic acid in the t est samples was determined by high performance liqu id chromatography with UV detection (HPLC/UV) using an external standard. The test item gave a chromatographic profile consisting of a sin gle peak.

Test Item The test item is described in the biological part o f this study.

Analytical Standard The test item described in the biological part of t his study was also used as the analytical standard.

Analytical Procedure

Storage The samples were stored at approximately -20 °C pri or to analysis.

Reagents and Solvents

Water: prepared using an ELGA Pure Lab Option R-15 water purification system.

Test medium: as described in the biological part of this study.

Solvents: acetonitrile, Fisher Scientific, HPLC gra de. tetrahydrofuran, Sigma-Aldrich, HPLC grade.

Reagents: tetrabutyl ammonium hydroxide, Fisher Sci entific, HPLC grade.

Preparation of Calibration Standards The test item (nominal 500 mg) was dissolved in tet rahydrofuran (100 mL) to prepare a stock solution w ith a nominal concentration of 5000 mg/L. This stock solution was further diluted with water to obtain a nominal 100 mg/L calibration stan dard. A duplicate calibration standard was similarly prepared at 100 mg/L. These duplicate calibration standards were used to determine the r ecovery and test sample concentrations.

Preparation of Linearity Standards The test item (nominal 500 mg) was dissolved in tet rahydrofuran (100 mL) to prepare a stock solution w ith a nominal concentration of 5000 mg/L. Defined volumes of this stock solution were d iluted with water to obtain standards in the range of 1.0 to 150 mg/L. A second standard was similarly prepared at a nominal concentration o f 100 mg/L. These standards were used to evaluate t he linearity of the analytical system.

Preparation of Spiked Recovery Samples To demonstrate the validity of the analytical proce dure, volumes of test medium were spiked with the t est item and its recovery was assessed. The test item (nominal 500 mg) was initially dissol ved in tetrahydrofuran to prepare a stock solution with a concentration of 10000 mg/L. This stock solution was further diluted with tetrahydrof uran to produce another solution of 625 mg/L. Defin ed volumes of both of these stock solutions were diluted with test medium to obtain s piked recovery samples at concentrations of 100 mg/ L and 6.25 mg/L. Five replicates at each concentration level were prepared and subjected to the same treat ment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

Preparation of Test Samples The test samples were thawed with the aid of sonica tion and then centrifuged (4500 rpm, 10 minutes) to remove algal cells, and then analyzed directly using the instrumental procedure. 

# Page 2 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

### Vehicle

Details on test solutions



## Test organisms
Test organisms (species)

Details on test organisms Instrumental Parameters

Autosampler: HP 1260 Series Pump: HP 1260 Series Detector: HP 1260 Series Column oven: HP 1260 Series

Column: Kinetex XB-C18; 50 mm x 3.0 mm; 2.6 μm

Mobile phase: water: acetonitrile:0.1M tetrabutylam monium hydroxide (475:125:25, v/v/v) Injection volume: 10 μ L

Flow rate: 0.5 mL/minute

Column temperature: 30 °C

Detection wavelength: 210 nm

Retention time: approximately 1.9 minutes

For full information pease see the attached Appendi x 4, Analytical Investigations

no

Range-finding test The test concentrations to be used in the initial e xperiment were determined by a preliminary range-fi nding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapita ta cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 96 hours.

The test was conducted in 250 mL glass conical flas ks each containing 100 mL of test preparation and p lugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test i tem was dissolved directly in culture medium.

An amount of test item (50 mg) was dissolved in cul ture medium and the volume adjusted to 500 mL to gi ve a 100 mg/L stock solution from which a series of dilutions was made to give furthe r stock solutions of 10, 1.0 and 0.10 mg/L. An aliq uot (450 mL) of each of the stock solutions was separately inoculated with 4.7 mL of algal suspensi on within 30 minutes of preparation to give the req uired nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

The stock solutions and each of the prepared concen trations were inverted several times to ensure adeq uate mixing and homogeneity.

The control group was maintained under identical co nditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the c ell density determined using a Coulter® Multisizer Particle Counter. The flasks we re then plugged with polyurethane foam bungs and in cubated (INFORS Multitron® Version 2 incubator) at 24 ± 2 oC under continuous illuminati on (intensity approximately 4300 lux) provided by w arm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 96 h ours.

After 96 hours the cell density of each flask was d etermined using a Coulter® Multisizer Particle Coun ter. A sample of each test concentration was taken for c hemical analysis at 0 and 96 hours in order to dete rmine the stability of the test item under test conditions. All samples were stored at approximatel y -20 °C prior to analysis.

Initial experiment Based on the results of the range-finding test the following nominal test concentrations were assigned to the initial experiment: 6.25, 12.5, 25, 50 and 100 mg/L.

Experimental Preparation For the purpose of the initial experiment, the test item was dissolved directly in culture medium.

An amount of test item (100 mg) was dissolved in cu lture 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 furthe r 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 suspens ion within 30 minutes of preparation to give the re quired nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L.

The stock solutions and each of the prepared concen trations were inverted several times to ensure adeq uate mixing and homogeneity.

Pseudokirchnerella subcapitata 

# Page 3 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

## Study design
Test type

Water media type

Limit test

Total exposure duration

Post exposure observation period



## Test conditions

Hardness

Test temperature

pH

Dissolved oxygen

Salinity

Nominal and measured concentrations Test Species The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of P seudokirchneriella subcapitata were obtained from the Culture Collection of Algae and P rotozoa (CCAP), SAMS Research Services Ltd, Scottis h Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory b y the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant ill umination at 21 ± 1 °C.

Prior to the start of the test sufficient master cu lture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10 3 cells/mL. The flasks were plugged with polyuretha ne foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm ) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/mL.

A positive control (Harlan Study Number: 41200591) used zinc chloride as the reference item. Details o f the positive control are given in the attached Appendix 2.

Culture Medium The culture medium used for both the range-finding and definitive tests was the same as that used to m aintain the stock culture.

The culture medium is defined in the attached Appen dix 3.

static

freshwater

no

96 h

Not applicable

Not recorded.

Temperature was maintained at 24 ± 2 oC throughout the test. The temperature within the incubator was recorded hourly using the incubators’ internal pt-100 temperature probe and Multicom Soft ware Package (see attached Figure 2).

A concentration dependant decline in pH was observe d at 0 hours during the initial experiment in the r ange of pH 7.4 at 6.25 mg/L through to pH 2.5 at 100 mg/L. It was therefore considered tha t the inhibition of growth observed at the test con centrations 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.

The pH value of the control cultures (see Table 4) was observed to increase from pH 7.5 at 0 hours to pH 7.7 at 96 hours. The pH deviation in the control cultures was less than 1.5 pH units after 96 hours and therefore was within the limits given in the Te st Guidelines.

The 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 inc ubator was recorded hourly using the incubators’ in ternal pt-100 temperature probe and Multicom Software Package (see attached Figure 2).

Not recorded.

freshwater used

The range-finding test was conducted by exposing Ps eudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 96 hours. Based on the results of the initial experiment the following nominal test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 

# Page 4 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

### Details on test conditions

Reference substance (positive control)

Any other information on materials and methods incl . tables and 100 mg/L.

Exposure conditions As in the range-finding test and initial experiment 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three fla sks each containing 100 mL were used for each treat ment group.

The control group was maintained under identical co nditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.00 x 105 cells per mL. Inoculation of

500 mL of test medium with 10.0 mL of this algal su spension gave an initial nominal cell density of 1 x 104 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bung s and incubated (INFORS Multitron® Version 2 incuba tor) at 24 ± 2 °C under continuous illumination (intensity approximately 4300 lux) pro vided by warm white lighting (380 – 730 nm) and con stantly shaken at approximately 150 rpm for 96 hours.

Samples were taken at 0, 24, 46, 70 and 96 hours an d the cell densities determined using a Coulter® Mu ltisizer Particle Counter.

Culture Medium The culture medium used for both the range-finding and definitive tests was the same as that used to m aintain the stock culture.

The culture medium is defined in the attached Appen dix 3.

yes zinc chloride as the reference item at concentr ations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. (s ee attached Appendix 2)

Evaluation of data Comparison ofgrowthrates The average specific growth rate for a specified pe riod is calculated as the logarithmic increase in b iomass from the equation:


## μ = 1n N n 

## – 1n N 1 

## ÷ t n 

## - t 1

where:

μ = average specific growth rate fr om time t 1 to t n
N 1 = cell concentration at t 1

N n = cell concentration at t n

t 1 = time of first measurement

t n = time of n th measurement

The average specific growth rate over the test dura tion was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order t o increase the precision of the calculation.

In addition the section by section specific growth rate (days 0-1, 1-2, 2-3 and 3-4) was calculated fo r the control cultures and the results examined in order to determine whether the growth r ate remained constant.

Percentage inhibition of growth rate for each repli cate test item vessel was calculated using the foll owing equation: 

## I r 

## = μ c 

## - μ t 

## ÷ μ c 

## x 100

where:

I r = percentage inhibition of average specific growth rate

μ c = an average specific growth rate f or the control cultures

μ t = average specific growth rate for the test culture

ComparisonofYield Yield is calculated as the increase in biomass over the exposure period using the following equation:



## Y = N n 

## – N 0

where:

Y = yield

N 0 = cell concentration at the start of the test

N n = cell concentration at the end of t he test

For each test concentration and control the mean va lue for yield along with the standard deviation was calculated. Percentage inhibition of yield was calculated using the following equation: 

# Page 5 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

# Results and discussions

Effect concentrations 

## I y 

## = (Y c 

## - Y t 

## ) ÷ Y c 

## x 100

where:

I y = percentage inhibition of yield

Y c = mean value for yield in the contro l group

Y t = mean value for yield for the treat ment group

Comparison of biomass integral

The biomass integral (area under the growth curve) was calculated using the following equation:



## A = N 1 

## – N 0 

## ÷ 2 x t 1 

## + N 1 

## + N 2 

## – 2N 0 

## ÷ 2 x (t 2 

## – t 1 

## ) + N n-1 

## + N n 

## – 2N 0 

## ÷ 2 x (t n 

## – t n-1 

## )

where:

A = area

N 0 = nominal cell concentration at start of test

N 1 = measured cell concentration at t 1

N n = measured cell concentration at t n

t 1 = time of first measurement after b eginning of test

t n = time of n th measurement after beginning of test

Percentage inhibition of the biomass integral for e ach replicate test item vessel was calculated using the following equation:



## I A 

## = A c 

## - A t 

## ÷ A c 

## x 100

where:

I A = percentage inhibition of the bioma ss integral

A c = mean biomass integral for the cont rol cultures

A t = biomass integral for the test cult ure

Determination of EC x values For each individual test vessel (mean values for yi eld), percentage inhibition (arithmetic axis) was p lotted against test concentration (logarithmic axis) and a line fitted by computerise d interpolation using the Xlfit software package (I DBS). EC x values were then determined from the equation for the fitted line.

It was not possible to calculate 95% confidence lim its for the EC 50 values as the data generated did not fit the models available for the

calculation of confidence limits.

Statisticalanalysis One way analysis of variance incorporating Bartlett 's test for homogeneity of variance (Sokal and Rohl f 1981) and Dunnett's multiple comparison procedure for comparing several treatmen ts with a control (Dunnett 1955) was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the control and all test concentrations to determine any stati stically significant differences between the test and control groups. All statistical analyses w ere performed using the SAS computer software packa ge (SAS 1999 - 2001).

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

The cell concentration 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 se ction by section specific growth rates in the contr ol cultures during the course of the test (0- 24, 24-48 and 48-72 hours) must not exceed 35% (OEC D Guidelines).

The coefficient of variation of the average specifi c growth rate in replicate control cultures after 7 2 hours must not exceed 7% (OECD Guidelines).

The coefficient of variation of the cell density va lues in replicate control cultures must not exceed 20% after 72 and 96 hours (EPA/OPPTS Guidelines). 

# Page 6 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

### Details on results Duration Endpoint Effect conc. Nominal/Measured Conc. based on Basis for effect Remarks (e.g. 95% CL)

96 h EC50 > 100 mg/L nominal test mat. cell nu mber

96 h NOEC 100 mg/L nominal test mat. cell numb er

RESULTS

Range-finding Test The cell densities and percentage inhibition of gro wth values from the exposure of Pseudokirchneriella subcapitata to the test item during the

range-finding test are given in Table 1.

The results showed no effect on growth at the nomin al test concentrations of 0.10, 1.0 and 10 mg/L. Ho wever, growth was observed to be reduced at 100 mg/L.

Based on this information nominal test concentratio ns of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the initial experiment.

Chemical analysis of the 10 and 100 mg/L test prepa rations at 0 and 96 hours (see attached Appendix 4) showed measured concentrations of the degradation product to range from 13 to 95 mg/L ind icating that the degradation product was stable ove r the test period.

Initial Experiment Cell density values determined at each sampling tim e and pH values at 0 and 96 hours are given in Tabl e 2.

A concentration dependant decline in pH was observe d at 0 hours during the initial experiment in the r ange of pH 7.4 at 6.25 mg/L through to pH 2.5 at 100 mg/L. It was therefore considered tha t the inhibition of growth observed at the test con centrations of 25, 50 and 100 mg/L may have been due to the acidic nature of the test item rather th an its true toxicity. As such it was considered app ropriate to perform the definitive test using pH adjusted test concentrations in order to determine the true toxic effect of the test item.

Based on the results of the initial experiment the following nominal test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/L.

Definitive Test Cell density values determined at each sampling tim e are given in Table 3. pH values pre and post adju stment at 0 hours and following 96 hours exposure are given in Table 4. Daily specific growt h rates for the control cultures are given in Table 5. Growth rate, yield and biomass integral values for the control and test cultures after 72 h ours and percentage inhibition values are given in Table 6. Growth rate, yield and biomass integral values for the control and test cultures a fter 96 hours and percentage inhibition values are given in Table 7.

The mean cell densities versus time for the definit ive test are presented in the attached Figure 1.

Verification of test concentrations The test item was known to rapidly hydrolyse in wat er (half-life less than 5 minutes) forming benzenes ulfonic acid. In accordance with the OECD Guidance Document on Aquatic Testing of Difficult S ubstances and Mixtures (2000) as the half life was less than 1-Hour, analysis of the test preparations was performed for the hydrolysis produ ct only. Analysis of the 100 mg/L test preparations at 0 and 96 hours (see the attached Appendix 5) showed measured concentrations of the h ydrolysis product to range from 104 to 106 mg/L.

Validation criteria The following data show that the cell concentration of the control cultures increased by a factor of 8 0 after 72 hours and by a factor of 272 after 96 hours. This increase was in line with the OECD G uideline that states the enhancement must be at lea st by a factor of 16 after 72 hours and the EPA/OPPTS Guidelines that states the enhancement mu st be at least by a factor of 100 after 96 hours.

Mean cell density of control at 0 hours : 8.45 x 10 E+3 cells per mL Mean cell density of control at 72 hours : 6.76 x 1 0E+5 cells per mL Mean cell density of control at 96 hours : 2.30 x 1 0E+6 cells per mL

The mean coefficient of variation for section by se ction specific growth rate for the control cultures over the test period (0-72 hours) was 16% and hence satisfied the validation criterion given in t he OECD Guideline which states the mean must not ex ceed 35%.

The coefficient of variation for average specific g rowth rate for the control cultures over the test p eriod (0 - 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline wh ich states that this must not exceed 7%.

The coefficient of variation of the cell density va lues in replicate control cultures was 16% after 72 hours and 5% after 96 hours and hence satisfied the validation criterion which states that this mus t not exceed 20%.

Growth data From the data given in Tables 3, 6 and 7, it is cle ar that the growth rate (r), yield (y) and biomass integral (b) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of t he degradation product over the 96-Hour exposure pe riod. 

# Page 7 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

### Results with reference substance (positive control) Accordingly the following results were determined f rom the data:

Inhibition of growth rate

ErC10 (0 - 72 h) : >100 mg/L ErC20 (0 - 72 h) : >100 mg/L ErC50 (0 - 72 h) : >100 mg/L ErC10 (0 - 96 h) : >100 mg/L ErC20 (0 - 96 h) : >100 mg/L ErC50 (0 - 96 h) : >100 mg/L

where ErCx is the test concentration that reduced g rowth rate by x%.

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and al l test concentrations using one way analysis of variance incorporating Bartlett's test for homogene ity of variance (Sokal and Rohlf 1981) and Dunnett' s multiple comparison procedure for comparing several treatments with a control (Dunnet t 1955). There were no statistically significant de creases in growth rate (P ≥ 0.05), between the control and all test groups and therefore the "No O bserved Effect Concentration" (NOEC) based on growt h rate was 100 mg/L.

Inhibition of yield

EyC10 (0 - 72 h) : >100 mg/L EyC20 (0 - 72 h) : >100 mg/L EyC50 (0 - 72 h) : >100 mg/L EyC10 (0 - 96 h) : >100 mg/L EyC20 (0 - 96 h) : >100 mg/L EyC50 (0 - 96 h) : >100 mg/L

where EyCx is the test concentration that reduced y ield by x%.

Statistical analysis of the yield data at 72 and 96 hours was carried out as in Section 4.3.3.1. There were no statistically significant decreases in yield (P ≥ 0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (N OEC) based on yield was 100 mg/L.

Inhibition of biomass integral

EbC10 (0 - 72 h) : >100 mg/L EbC20 (0 - 72 h) : >100 mg/L EbC50 (0 - 72 h) : >100 mg/L EbC10 (0 - 96 h) : >100 mg/L EbC20 (0 - 96 h) : >100 mg/L EbC50 (0 - 96 h) : >100 mg/L

where EbCx is the test concentration that reduced b iomass integral (area under the growth curve) by x%

.
Statistical analysis of the biomass integral data a t 72 and 96 hours was carried out. There were no st atistically significant decreases in biomass integral (P ≥ 0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (N OEC) based on biomass integral was 100 mg/L.

Observations on cultures All test and control cultures were inspected micros copically at 96 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test item solubility At the start of the test all control and test cultu res were observed to be clear colorless solutions. After the 96-Hour test period all control and
test cultures were observed to be bright green dispersio ns.

Physico-chemical measurements The pH values of the control and each test preparat ion are given in Table 4. Temperature was maintaine d at 24 ± 2 oC throughout the test.

The pH value of the control cultures (see Table 4) was observed to increase from pH 7.5 at 0 hours to pH 7.7 at 96 hours. The pH deviation in the control cultures was less than 1.5 pH units after 9 6 hours and therefore was within the limits given i n the Test Guidelines.

Positive Control (see attached Appendix 2) A positive control (Harlan Study Number: 41200591) used zinc chloride as the reference item at concent rations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.

Exposure conditions and data evaluation for the pos itive control were similar to those in the definiti ve test.

Exposure of Pseudokirchneriella subcapitata (CCAP 2 78/4) to the reference item gave the following resu lts:

Time Point(Hours) Response Variable EC50 (mg/L) 95% Confidence Limits (mg/L) No Observed Effect Lowest Observed Effect Concentration (NOEC) (mg/L) Concentration (LOEC) (m g/L) 72 Growth Rate 0.31 0.28 - 0.35 0.10 0.32 Yield 0.26 * 0.10 0.32 

# Page 8 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

### Reported statistics and error estimates

Any other information on results incl. tables Biomass 0.27 * 0.10 0.32 96 Growth Rate 0.35 0.30 - 0.40 0.10 0.32 Yield 0.27 * 0.10 0.32 Biomass 0.26 * 0.10 0.32

The results from the positive control with zinc chl oride were within the normal ranges for this refere nce item.

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and al l test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of va riance (Sokal and Rohlf 1981) and Dunnett's multipl e comparison procedure for comparing several treatments with a control (Dunnett 1955).

There were no statistically significant decreases i n growth rate (P ≥ 0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/L.

There were no statistically significant decreases i n yield (P ≥ 0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 mg/L.

There were no statistically significant decreases i n biomass integral (P ≥ 0.05), between the control and all test groups and therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 100 mg/L.

TABLES Table 1 Preparation of Test Samples

- 

### = not applicable

Table 2 Linearity Data

Table 3 Results for Range-Finding Samples

Nominal

Concentration of Test Item Sample Volume Final Volume Sample Preparation Factor Final Nominal Concentration

of Degradation Product

[mg/L] [mL] [mL] F [mg/L] Control 100 100 1.0

- 100 100 100 1.0 100

Concentration of Degradation Product Area

[mg/L] [counts]
0 0
0.998 8.625 x 10 5

4.99 4.398 x 10 6

9.98 8.772 x 10 6

20.0 1.744 x 10 7

49.9 4.385 x 10 7

99.8 8.475 x 10 7

102 8.678 x 10 7

150 1.240 x 10 8

Time point Nominal Concentration of

Test Item in RangeFinding Sample

c nom Measured

Concentration of Degradation Product in Sample Vial

x Sample Preparation Factor

F Determined

Concentration of DegradationProduct in RangeFinding Sample

c

[hours] [mg/L] [mg/L] [mg/L]

0 10 13.1 1.0 13.1

100 95.4 1.0 95.4

96 10 13.4 1.0 13.4 

# Page 9 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition T

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

# Overall remarks, attachments

Remarks on results including tables and figures Table 4 Results for Spiked Recovery Samples

For reporting purposes, x has been calculated from c retrospectively. Table 5 Results for Test Samples

LOQ 

### = Limit of Quantitation For reporting purposes, x has been calculated from c retrospectively. 100 97.4 1.0 97.4

Nominal Concentration of

Test Item

c nom Fortified

Concentration of Test Item in the Spiked Sample

c fort Measured

Concentration of Degradation Product in the Sample Vial

x Sample

Preparation Factor

F Determined

Concentration of Degradation Product in the Spiked Sample

c Analytical

Recovery

Rate

R Precision

(Relative Standard Deviation of Recovery)

[mg/L] [mg/L] [mg/L] [mg/L] [%] [%]

6.25 6.35 6.57 1.0 6.57

104 0.23

6.35 6.58 1.0 6.58

6.35 6.61 1.0 6.61

6.35 6.59 1.0 6.59

6.35 6.59 1.0 6.59

100 102 102 1.0 102

100 0.144

102 102 1.0 102

102 102 1.0 102

102 102 1.0 102

102 102 1.0 102

Acceptance Target 80

- 120 <10

Time point Nominal

Concentration of

Test Item in Test Sample

c nom Measured

Concentration



### of Degradation Product in Sample Vial

x Sample

Preparation Factor

F Determined

Concentration of Degradation Product in Test Sample

c

[hours] [mg/L] [mg/L] [mg/L]

0 Control <LOQ 1.0 <LOQ

100 104 1.0 104

96 Control <LOQ 1.0 <LOQ

100 106 1.0 106

FIGURES Figure 1..........Mean Cell Densities v Time for th e Definitive Test Figure 2..........Temperature Measurements Taken Th roughout the Test Period APPENDICES Appendix 1....Certificate of Analysis Appendix 2....Positive Control Appendix 3....Culture Medium Appendix 4....Analytical Investigations

REFERENCES DUNNETT, C W (1955) A Multiple Comparison Procedure for Comparing Several Treatments With a Control. J Am Stat Assoc 50 , 1096- 1121.

Environment Directorate, Organisation for Economic Co-operation and Development (OECD) (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtur es. 

# Page 10 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...

### Attached background material



# Applicant's summary and conclusion

Validity criteria fulfilled

Conclusions

Executive summary SAS/STAT Proprietary Software Release 8.02 (1999 - 2001), SAS Institute Inc, Cary, NC, USA.

SOKAL, R R and ROHLF, F J (1981) Biometry.New York: W H FREEMAN and Company.

Xlfit, ID Business Solutions Ltd.

Attached document Remarks

Figures 1 & 2 Algal Inhibition Test Benzenesulfonyl chloride Lanxess Corporation 41201584.pdf / 71.6 K B (application/pdf) Figures 1 & 2

Appendices Algal Inhibition Test Benzenesulfonyl ch loride Lanxess Corporation 41201584.pdf / 892.28 KB (application/pdf) Appendices

yes

CONCLUSION The effect of the degradation product on the growth of Pseudokirchneriella subcapitata has been invest igated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Conce ntration was 100 mg/L.

Introduction A study was performed to assess the effect of the t est item on the growth of the green alga Pseudokirchneriella subcapitata . The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alg a and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commis sion Regulation (EC) No 761/2009, the US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400 and ASTM E1218-04.

Methods Following a preliminary range-finding test and init ial experiment, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L (three replicate flasks per con centration) for 96 hours, under constant illumination and shaking at a temperature of 24 ± 2 °C. Due to the extremely acidic nature of the test item, the pH of the test preparations was adjusted to 7.5 prior to the addition of the al gal cells at the start of the test.

Samples of the algal populations were removed daily and cell concentrations determined for each contro l and treatment group, using a
Coulter ® Multisizer Particle Counter.

Results The test item was known to rapidly hydrolyse in wat er (half-life less than 5 minutes) forming benzenes ulfonic acid. In accordance with the OECD Guidance Document on Aquatic Testing of Diffic ult Substances and Mixtures (2000) as the half life was less than 1-Hour, analysis of the test preparations was performed for the hydroly sis product only. Analysis of the 100 mg/L test pre parations at 0 and 96 hours showed measured concentrations of the hydrolysis product t o range from 104 to 106 mg/L.

Exposure of Pseudokirchneriella subcapitata to the degradation product gave EC 50 values of greater than 100 mg/L. The No Observed Ef fect Concentration was 100 mg/L. 

# Page 11 of 11 Endpoint study record: Toxicity to aquatic algae an d cyanobacteria Algal Inhibition

...

25/03/2013 file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chlor ide\Lanxess

- Benzenesulfonyl

...