Endpoint study record: Toxicity to aquatic algae and 

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 Experimental 

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 the 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 

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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 - R3 pooled) at 0 and 96 hours for 

quantitative analysis. All samples were stored at approximately -20 °C prior to analysis. Duplicate samples 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 were analyzed. 

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

The test item is known to hydrolyse quickly in the presence of water to the corresponding benzenesulphonic acid (half-life of approximately 

5 minutes). It is understood that the peak observed in the chromatograms was not the original benzenesulphonyl chloride but was actually 

benzenesulphonic acid, which is the stable degradation product. 

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. 

Test Item 

The test item is described in the biological part of this study. 

Analytical Standard 

The test item described in the biological part of this study was also used as the analytical standard.

Analytical Procedure 

Storage 

The samples were stored at approximately -20 °C prior 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 grade. 

tetrahydrofuran, Sigma-Aldrich, HPLC grade. 

Reagents: tetrabutyl ammonium hydroxide, Fisher Scientific, HPLC grade. 

Preparation of Calibration Standards 

The test item (nominal 500 mg) was dissolved in tetrahydrofuran (100 mL) to prepare a stock solution with a nominal concentration of 5000 

mg/L. This stock solution was further diluted with water to obtain a nominal 100 mg/L calibration standard. A duplicate calibration standard 

was similarly prepared at 100 mg/L. These duplicate calibration standards were used to determine the recovery and test sample 

concentrations. 

Preparation of Linearity Standards 

The test item (nominal 500 mg) was dissolved in tetrahydrofuran (100 mL) to prepare a stock solution with a nominal concentration of 5000 

mg/L. Defined volumes of this stock solution were diluted 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 of 100 mg/L. These standards were used to evaluate the linearity of the analytical system.

Preparation of Spiked Recovery Samples 

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and its recovery was 

assessed. 

The test item (nominal 500 mg) was initially dissolved in tetrahydrofuran to prepare a stock solution with a concentration of 10000 mg/L. This 

stock solution was further diluted with tetrahydrofuran to produce another solution of 625 mg/L. Defined volumes of both of these stock 

solutions were diluted with test medium to obtain spiked 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 treatment 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 sonication and then centrifuged (4500 rpm, 10 minutes) to remove algal cells, and then 

analyzed 

directly using the instrumental procedure. 

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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 tetrabutylammonium hydroxide (475:125:25, v/v/v) 

Injection volume: 10 μL 

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 Appendix 4, Analytical Investigations 

no 

Range-finding test 

The test concentrations to be used in the initial experiment were determined by a preliminary range-finding test. The range-finding test was 

conducted by exposing Pseudokirchneriella 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. 

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam 

bungs to 

reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture 

medium. 

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 to give the required nominal test concentrations of 

0.10, 1.0, 10 and 100 mg/L. 

The stock solutions and 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. 

At the start of the range-finding test a sample of each test and control culture was removed and the cell density 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 oC 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. 

After 96 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. 

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. All samples were stored at approximately -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 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. 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 to give the required nominal test concentrations of 

6.25, 12.5, 25, 50 and 100 mg/L. 

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. 

Pseudokirchnerella subcapitata 

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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 Pseudokirchneriella subcapitata were 

obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, 

Scotland. 

Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in 

the 

laboratory under constant aeration and constant illumination at 21 ± 1 °C. 

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks 

to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane 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 of 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 maintain the stock culture. 

The culture medium is defined in the attached Appendix 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 Software Package (see attached Figure 2). 

A concentration dependant 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. 

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 Test 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 incubator was recorded hourly using the incubators’ internal pt-100 temperature probe 

and Multicom 

Software Package (see attached Figure 2). 

Not recorded.

freshwater used

The range-finding test was conducted by exposing Pseudokirchneriella 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 

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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 flasks each containing 100 mL were used for each treatment group. 

The control group was maintained under identical conditions 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 suspension 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 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. 

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

Culture Medium 

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

The culture medium is defined in the attached Appendix 3. 

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

Evaluation of data

Comparison ofgrowthrates

The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

μ = 1n Nn– 1n N1 ÷ tn - t1

where:

μ = average specific growth rate from time t1to tn

N1 = cell concentration at t1

Nn = cell concentration at tn

t

1

 = time of first measurement

t

n

 = time of nthmeasurement

The average specific growth rate over the test duration 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 to 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 for the control cultures and the results 

examined in order to determine whether the growth rate remained constant.

Percentage inhibition of growth rate for each replicate test item vessel was calculated using the following equation:

I

r

 = μc- μt ÷ μc x 100 

where:

I

r

 = percentage inhibition of average specific growth rate

μc = an average specific growth rate for the control cultures

μt = 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 = Nn

– N0

where:

Y = yield

N0 = cell concentration at the start of the test

Nn = cell concentration at the end of the test

For each test concentration and control the mean value for yield along with the standard deviation was calculated. Percentage inhibition of 

yield was calculated using the following equation:

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Results and discussions 

Effect concentrations 

I

y

= (Yc- Yt) ÷ Yc x 100

where:

I

y

 = percentage inhibition of yield

Yc = mean value for yield in the control group

Yt = mean value for yield for the treatment group

Comparison of biomass integral

 

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

A = N1– N0 ÷ 2 x t1+ N1+ N2– 2N0÷ 2 x (t2– t1) + Nn-1+ Nn– 2N0÷ 2 x (tn– tn-1) 

where:

A = area

N0 = nominal cell concentration at start of test

N1 = measured cell concentration at t1

Nn = measured cell concentration at tn

t

1

 = time of first measurement after beginning of test

t

n

 = time of nthmeasurement after beginning of test

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

IA= Ac- At÷ Ac x 100 

where:

IA = percentage inhibition of the biomass integral

Ac = mean biomass integral for the control cultures

At = biomass integral for the test culture

Determination of ECxvalues

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration 

(logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECxvalues were then determined 

from the equation for the fitted line.

It was not possible to calculate 95% confidence limits for the EC50values 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 Rohlf 1981) and Dunnett's multiple 

comparison procedure for comparing several treatments 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 statistically significant differences between the 

test and control groups. All statistical analyses were performed using the SAS computer software package (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 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).

 

 

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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 number 

 

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

 

RESULTS 

Range-finding Test 

The cell densities and percentage inhibition of growth 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 nominal test concentrations of 0.10, 1.0 and 10 mg/L. However, growth was observed to be 

reduced at 100 mg/L. 

Based on this information nominal test concentrations 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 preparations at 0 and 96 hours (see attached Appendix 4) showed measured concentrations 

of the 

degradation product to range from 13 to 95 mg/L indicating that the degradation product was stable over the test period. 

Initial Experiment 

Cell density values determined at each sampling time and pH values at 0 and 96 hours are given in Table 2. 

A concentration dependant 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. 

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 time are given in Table 3. pH values pre and post adjustment at 0 hours and following 96 

hours 

exposure are given in Table 4. Daily specific growth 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 hours and percentage inhibition values are given in Table 6. Growth rate, yield and biomass 

integral values for the control and test cultures after 96 hours and percentage inhibition values are given in Table 7. 

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

Verification of test concentrations 

The test item was known to rapidly hydrolyse in water (half-life less than 5 minutes) forming benzenesulfonic acid. In accordance with the 

OECD 

Guidance Document on Aquatic Testing of Difficult Substances and Mixtures (2000) as the half life was less than 1-Hour, analysis of the test 

preparations was performed for the hydrolysis product only. Analysis of the 100 mg/L test preparations at 0 and 96 hours (see the attached 

Appendix 5) showed measured concentrations of the hydrolysis 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 80 after 72 hours and by a factor of 272 

after 

96 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours 

and the 

EPA/OPPTS Guidelines that states the enhancement must be at least by a factor of 100 after 96 hours. 

Mean cell density of control at 0 hours : 8.45 x 10E+3 cells per mL 

Mean cell density of control at 72 hours : 6.76 x 10E+5 cells per mL 

Mean cell density of control at 96 hours : 2.30 x 10E+6 cells per mL 

The mean coefficient of variation for section by section specific growth rate for the control cultures over the test period (0-72 hours) was 16% 

and 

hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. 

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 3% and hence satisfied 

the 

validation criterion given in the OECD Guideline which states that this must not exceed 7%. 

The coefficient of variation of the cell density values in replicate control cultures was 16% after 72 hours and 5% after 96 hours and hence 

satisfied 

the validation criterion which states that this must not exceed 20%. 

Growth data 

From the data given in Tables 3, 6 and 7, it is clear 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. 

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Results with reference substance (positive control) 

Accordingly the following results were determined from 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 growth rate by x%. 

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and all test concentrations using one way 

analysis of 

variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for 

comparing several treatments with a control (Dunnett 1955). There were no statistically significant decreases in 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. 

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 yield 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" (NOEC) 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 biomass integral (area under the growth curve) by x%. 

Statistical analysis of the biomass integral data at 72 and 96 hours was carried out. There were no statistically significant decreases in 

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. 

Observations on cultures 

All test and control cultures were inspected microscopically 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 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. 

Physico-chemical measurements 

The pH values of the control and each test preparation are given in Table 4. Temperature was maintained 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 96 hours and therefore was within the limits given in the Test Guidelines. 

Positive Control (see attached Appendix 2) 

A positive control (Harlan Study Number: 41200591) used zinc chloride as the reference item at concentrations of 0.010, 0.032, 0.10, 0.32 

and 

1.0 mg/L. 

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. 

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results: 

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) (mg/L) 

72 Growth Rate 0.31 0.28 - 0.35 0.10 0.32 

Yield 0.26 * 0.10 0.32 

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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 chloride were within the normal ranges for this reference item. 

Statistical analysis of the growth rate data at 72 and 96 hours was carried out for the control and all test concentrations using one way 

analysis of variance 

incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing 

several 

treatments with a control (Dunnett 1955). 

There were no statistically significant decreases in 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 in 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 in 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 105

4.99 4.398 x 106

9.98 8.772 x 106

20.0 1.744 x 107

49.9 4.385 x 107

99.8 8.475 x 107

102 8.678 x 107

150 1.240 x 108

Time point

Nominal 

Concentration of 

Test Item in RangeFinding Sample 

cnom

Measured 

Concentration of 

Degradation 

Product in Sample 

Vial 

x

Sample Preparation 

Factor 

F

Determined 

Concentration of 

Degradation 

Product 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

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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 

cnom

Fortified 

Concentration

of Test Item in

the Spiked

Sample

cfort

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 

cnom

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 the Definitive Test 

Figure 2..........Temperature Measurements Taken Throughout 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 Assoc50, 1096-

1121. 

 

Environment Directorate, Organisation for Economic Co-operation and Development (OECD) (2000) Guidance Document on Aquatic 

Toxicity Testing of Difficult Substances and Mixtures. 

 

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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 KB (application/pdf) Figures 1 & 2 

Appendices Algal Inhibition Test Benzenesulfonyl chloride 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 investigated and gave EC50 values of 

greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. 

Introduction 

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella subcapitata. The method 

followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth 

Inhibition Test" referenced as Method C.3 of Commission 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 initial experiment,Pseudokirchneriella subcapitatawas 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 concentration) 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 algal cells at the start of the test. 

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a 

Coulter®Multisizer Particle Counter.

 

 

Results 

The test item was known to rapidly hydrolyse in water (half-life less than 5 minutes) forming benzenesulfonic acid. In accordance with the 

OECD Guidance Document on Aquatic Testing of Difficult Substances and Mixtures (2000) as the half life was less than 1-Hour, analysis of 

the test preparations was performed for the hydrolysis product only. Analysis of the 100 mg/L test preparations at 0 and 96 hours showed 

measured concentrations of the hydrolysis product to range from 104 to 106 mg/L. 

 

Exposure ofPseudokirchneriella subcapitatato the degradation product gave EC50values of greater than 100 mg/L. The No Observed Effect 

Concentration was 100 mg/L. 

 

 

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