Endpoint study record: Short-term toxicity to aquatic invertebrates Daphnia sp 

48-Hour Acute Immobilization Test Benzenesulfonyl chloride Lanxess 

Corporation 41201583 

Administrative Data 

Data source 

Reference 

Data access 

Data protection claimed 

Materials and methods 

Test guideline 

GLP compliance 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

Test material identity 

Test material form 

Details on test material 

Analytical monitoring 

Details on sampling 

UUID 

 

IUC5-6c94bbd5-5960-46a8-bbb7-924b947c4833 

Dossier UUID 

 

0 

Author 

 

admin4 / Harlan Laboratories Ltd. / Other / other: 

Date 

 

2013-03-21 08:07:27 GMT 

Remarks 

 

 

Purpose flag robust study summary 

Study result type experimental result Study period Experimental Starting Date: 10 September 2012 Experimental Completion Date: 05 March 

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 Daphnia sp., 48-Hour 

Acute Immobilization 

Test, Benzenesulfonyl 

chloride 

 

Harlan Laboratories 

Ltd. Shardlow Business 

Park Shardlow 

Derbyshire DE72 2GD 

UK 

41201583 Lanxess Corporation 111 

RIDC Park West Drive 

Pittsburgh PA 15275-1112 

UNITED STATES OF 

AMERICA 

 

2013-

03-19 

data submitter is data owner 

yes, but willing to share 

Qualifier Guideline Deviations 

according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test) no 

according to EU Method C.2 (Acute Toxicity for Daphnia) no 

according to EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids) no 

according to other guideline: ASTME729-96 no 

yes (incl. certificate) 

yes 

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 

supplied 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 

Storage conditions: room temperature in the dark 

yes 

Water samples were taken from the control and each test group (replicates R1 – R4 pooled) at 0 and 48 hours for quantitative analysis. All samples 

were stored at approximately -20 °C prior to analysis. 

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

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Details on analytical methods 

Vehicle 

Details on test solutions 

The method of analysis, recovery and test preparation analyses are described in the attched Appendix 5.

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, Fisher Scientific, HPLC grade. 

Reagents: tetrabutyl ammonium hydroxide 0.1M, 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 stocksolution was further 

diluted with tetrahydrofuran to produce another solution of 100 mg/L. Defined volumes of both of these stock solutions were diluted with test medium to obtain spiked 

recovery samples at concentrations of 1.0 mg/L and 100 mg/L. Five replicates at each concentration levelwere 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 analyzed directly using the instrumental procedure. 

Instrumental Parameters 

Autosampler: HP 1260 Series 

Pump: HP 1260 Series 

Detector: HP 1260 Series 

Column oven: HP 1260 Series 

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

Mobile phase: water:acetonitrile: 0.1M tetrabutyl ammonium hydroxide (buffered to pH 7) (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 2 minutes 

For full information please see attached Appendix 5, Analytical Investigations 

no 

Range-finding Test 

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

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L. The test item was 

dissolved directly in water. 

An amount of test item (50 mg) was dissolved in reconstituted water and the volume adjusted to 500 mL to give the 100 mg/L test concentration 

from which a series of dilutions was made to give further test concentrations of 10, 1.0 and 0.10 mg/L. 

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. 

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20 °C to 21 °C with a photoperiod 

of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL 

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Test organisms 

Test organisms (species) 

Details on test organisms 

Study design 

Test type 

Water media type 

Limit test 

Total exposure duration 

Post exposure observation period 

of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized 

Daphnia magna were recorded. 

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

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test 

conditions. 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: 10, 18, 32, 56 and 

100 mg/L. 

Experimental Preparation 

For the purpose of the initial experiment the test item was dissolved directly in reconstituted water.

An amount of test item (250 mg) was dissolved 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. 

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. 

Exposure Conditions 

In the initial experiment 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were 

placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels 

were then covered to reduce evaporation and maintained in a temperature controlled room at 20 °C to 22 °C with a photoperiod of 16 hours light 

(622 to 650 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure 

and the test vessels were not aerated. 

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

The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the 

start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after 

gentle agitation. 

Physico-Chemical Measurements 

Water 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. 

Definitive Test 

A concentration dependant decline in pH was observed at 0 hours during the initial experiment in the range of pH 7.2 – 7.4 at 10 mg/L through to 

pH 2.8 – 2.9 at 100 mg/L. It was therefore considered that the immobilization observed at the test concentrations of 56 and 100 mg/L may have 

been due to the acidic nature of the test item rather than its true toxicity. As such it was considered appropriate to perform the definitive test using pH adjusted test 

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

Due to the high water solubility of the test item and the stable nature of the hydrolysis product the use of a flow-through system of preparation was considered 

inappropriate. 

Based on the results of the initial experiment the following nominal test concentrations were assigned to the definitive test: 32, 56 and 100 mg/L. 

Experimental Preparation 

For the purpose of the definitive test the test item was dissolved directly in reconstituted water. 

An amount of test item (200 mg) was dissolved in reconstituted water and the volume adjusted to 2 liters to give the 100 mg/L test concentration 

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

adjusted to pH 8.0 prior to the addition of the daphnids. 

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. 

Daphnia magna 

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures. 

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see attached Appendix 2) in a temperature controlled room at approximately 20 

°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily 

with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by 

parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced were approximately 24 hours old. These 

young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or 

outcome of the study. 

A positive control (Harlan Study Number: 41203341) used potassium dichromate as the reference item. Details of the positive control are given in 

the attached Appendix 3. 

static 

freshwater 

no 

48 h 

Not applicable

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Test conditions 

Hardness 

Test temperature 

pH 

Dissolved oxygen 

Salinity 

Nominal and measured concentrations 

Details on test conditions 

Reference substance (positive control) 

Any other information on materials and methods incl. tables 

Results and discussions 

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

Temperature was maintained at 20 °C to 21 °C throughout the test. 

Due to a technical oversight the temperature in a single vessel was not logged hourly as required by the Test Guideline. This was considered to have had no 

adverse effect on the outcome of the test given that temperature measurements taken from each of the individual test vessels over the 48-Hour test period

showed that temperature remained within the guideline specification. 

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was

approximately air-saturation value.. 

The pH was measured using a Hach HQ30d Flexi handheld meter 

See attached Appendix 4 for results. 

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value. 

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen 

meter. 

See attached Appendix 4 for results. 

freshwater used.

Based on the results of the range-finding test the following nominal test concentrations were assigned to the initial experiment: 10, 18, 32, 56 and

100 mg/L. 

Based on the results of the initial experiment the following nominal test concentrations were assigned to the definitive test: 32, 56 and 100 mg/L. 

Exposure Conditions 

In the definitive test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were 

placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels 

were then covered to reduce evaporation and maintained in a temperature controlled room at 20 °C to 21 °C with a photoperiod of 16 hours light 

(633 to 656 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure 

and the test vessels were not aerated. 

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

The test preparations were not renewed during the exposure period. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the 

start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after 

gentle agitation. 

Physico-Chemical Measurements 

Water 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. 

yes (Potassium dichromate) 

Calculations 

A linear response was obtained during the validation of the analytical method, so for the definitive test samples a single-point calibration was performed using duplicate 

calibration standards. The mean peak areas of each calibration standard were corrected to a nominal concentration and the mean value taken. 

 

The concentration “x” of the degradation product in an injected sample was calculated by the following equation: 

 

 

 

The concentration of the degradation product in the test samples was calculated using the following equation: 

 

 

 

The recovery rate (R) of the spiked recovery samples was calculated using the following equation: 

 

 

X = Rspl. N ÷ Rcal std (1)

where x: concentration of the degradation product in injected sample [mg/L]

Rspl:

mean peak area response of the degradation product in injected sample [counts]

Rcal std:

mean peak area response for the calibration standard, corrected to nominal concentration [counts]

N: nominal calibration standard concentration [mg/L]

c = x ÷ R/100 . F (2)

where c: concentration of the degradation product in the test sample [mg/L]

x: concentration of thedegradation productin the injected sample solution [mg/L]

F:

R:

sample preparation factor recovery rate [%] (If not correcting for recovery then R is taken as 100)

R = c ÷ cfort . 100 (3)

where R: recovery rate [%]

c: determined concentration of thedegradation productin the spiked recovery sample [mg/L]

c

fort:

fortified concentration of thedegradation productin the spiked recovery sample [mg/L]

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Effect concentrations 

Details on results 

Results with reference substance 

Reported statistics and error estimates 

Any other information on results incl. tables 

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

24 h EC50 0.75 mg/L nominal test mat. mortality 95 % Confidence limits (mg/L) 0.56 - 1.0 

48 h EC50 0.45 mg/L nominal test mat. mortality 95% Confidence limits (mg/L) 0.42 - 0.48 

RESULTS 

Range-finding Test 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1. 

No immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However, immobilization was observed at 100 mg/L. 

Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the initial experiment. 

Chemical analysis of the 10 and 100 mg/L test preparations at 0 hours (see Appendix 5) showed measured degradation product concentrations of 14 and 138 mg/L 

respectively were obtained whilst concentrations of 14 and 145 mg/L respectively were obtained at 48 hours. These results 

indicated that the degradation product was stable over the test period. 

Initial Experiment 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. The results of 

the physico-chemical measurements are given in the attached Appendix 6. 

A concentration dependant decline in pH was observed at 0 hours during the initial experiment in the range of pH 7.2 – 7.4 at 10 mg/L through to 

pH 2.8 – 2.9 at 100 mg/L. It was therefore considered that the immobilization observed at the test concentrations of 56 and 100 mg/L may have 

been due to the acidic nature of the test item rather than its true toxicity. As such it was considered appropriate to perform the definitive test using pH adjusted test 

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

Based on the results of the initial experiment the following test concentrations were assigned to the definitive test: 32, 56 and 100 mg/L. 

Definitive Test 

Immobilization Data 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 3. 

Whilst a single daphnid was observed to be immobilized in each of the test preparations this was considered to be due to natural causes rather than a true toxic effect 

given that less than 10% immobilization occurred. The No Observed Effect Concentration after 24 and 48 hours exposure was 

100 mg/L. 

Observations on Test Item Solubility 

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

Physico-Chemical Measurements 

The results of the physico-chemical measurements are given in the attached Appendix 7. Temperature was maintained at 20 °C to 21 °C 

throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Due to a technical oversight the temperature in a single vessel was not logged hourly as required by the Test Guideline. This was considered to have had no adverse 

effect on the outcome of the test given that temperature measurements taken from each of the individual test vessels over the 

48-Hour test period showed that temperature remained within the guideline specification. 

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the 

presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the 

outcome or integrity of the test as no adverse effects were observed . 

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 preparation at 0 and 48 hours (see attached Appendix 5) showed measured 

hydrolysis product concentrations of 109 mg/L and 103 mg/L respectively were obtained. 

Positive Control 

A positive control (Harlan Study Number: 41203341) used potassium dichromate as the reference item at concentrations of 

0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. 

 

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

 

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 

48 hoursbased on the nominal test concentrations gave the following results: 

 

Time (h) EC50 (mg/L) 95% Confidence limits (mg/L) 

24 0.75 0.56 - 1.0 

48 0.45 0.42 - 0.48 

 

The No Observed Effect Concentrations after 24 and 48 hours were 0.56 and 0.32 mg/L respectively. The No Observed Effect Concentration is based 

upon zero immobilization at this concentration. 

 

The results from the positive control with potassium dichromate were within the normal range for this reference item. 

Evaluation of Data: An estimate of the EC50 values was given by inspection of the immobilization data. 

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at

48 hoursbased on the nominal test concentrations. 

TABLES

Table1 Cumulative Immobilization Data in the Range-finding Test 

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Table 2 Cumulative Immobilization Data in the Initial Experiment 

 

R1

– R4= Replicates 1 to 4 

Table 3 Cumulative Immobilization Data in the Definitive Test 

 

R1– R4= Replicates 1 to 4 

*Single immobilized daphnid considered to be due to natural causes rather than a true toxic effect as less than 10% immobilization occurred. 

ANALYTICAL TABLES

TABLES 

Table 1 Preparation of Test Samples 

 

- = not applicable

 

 

 

Table 2 Linearity Data 

 

Nominal

Concentration 

(mg/L)

Cumulative ImmobilizedDaphnia

(Initial Population: 10 Per Replicate)

 24 Hours 48 Hours

Control 0 0

0.10 0 0

1.0 0 0

10 0 0

100 10 10

Nominal

Concentration 

(mg/L)

Cumulative ImmobilizedDaphnia

(Initial Population: 5 Per Replicate)

24 Hours 48 Hours

R1

R2R3R4

Total % R1

R2R3R4

Total %

Control 0 0 0 0 0 0 0 0 0 0 0 0

10 0 0 0 0 0 0 0 0 0 0 0 0

18 0 0 0 0 0 0 0 0 0 0 0 0

32 0 0 0 0 0 0 0 0 0 0 0 0

56 3 4 5 5 17 85 5 5 5 5 20 100

100 5 5 5 5 20 100 5 5 5 5 20 100

Nominal

Concentration 

(mg/L)

Cumulative ImmobilizedDaphnia

(Initial Population: 5 Per Replicate)

24 Hours 48 Hours

R1

R2R3R4

Total % R1

R2R3R4

Total %

Control 0 0 0 0 0 0 0 0 0 0 0 0

32 0 0 0 0 0 0 1 0 0 0 1* 5

56 0 0 0 0 0 0 0 1 0 0 1* 5

100 1 0 0 0 1* 5 1 0 0 0 1* 5

Nominal 

Concentration of Test 

Item 

[mg/L]

Sample 

Volume 

[mL]

Final Volume

[mL]

Sample 

Preparation 

Factor 

F

Final Nominal 

Concentration of 

Degradation Product 

[mg/L]

Control 100 100 1.0 -

100 100 100 1.0 100

Concentration of Test Item 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

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Overall remarks, attachments 

Remarks on results including tables and figures 

 

Table 3 Results for Range-Finding Samples 

 

 

 

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

- = not applicable

For reporting purposes, x has been calculated from c retrospectively. 

 

 

Time point

Nominal 

Concentration of 

Test Item in 

Range-Finding 

Sample 

cnom

Measured 

Concentration 

of 

Degradation 

Product in 

Sample Vial 

x

Sample 

Preparation 

Factor 

F

Determined 

Concentration of 

Degradation 

Product in

Range-Finding 

Sample

c

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

0 10 14.0 1.0 14.0

100 138 1.0 138

48 10 13.8 1.0 13.8

100 145 1.0 145

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] [%] [%]

1.0 1.02 1.10 1.0 1.10

107 0.43

1.02 1.08 1.0 1.08

1.02 1.09 1.0 1.09

1.02 1.09 1.0 1.09

1.02 1.09 1.0 1.09

100 102 101 1.0 101

99 0.14

102 101 1.0 101

102 101 1.0 101

102 101 1.0 101

102 101 1.0 101

 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

100 109 1.0 109

48 Control <LOQ 1.0 <LOQ

100 103 1.0 103

Please see attached: 

APPENDICES 

Appendix 1....Certificate of Analysis 

Appendix 2....Reconstituted Water – Elendt M7 Medium 

Appendix 3....Positive Control 

Appendix 4....Reconstituted Water 

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Attached background material 

Applicant's summary and conclusion 

Validity criteria fulfilled 

Conclusions 

Executive summary 

Appendix 5....Analytical Investigations 

Appendix 6....Physico-Chemical Measurements from the Initial Experiment 

Appendix 7....Physico-Chemical Measurementsfrom the Definitive Test 

Appendix 8....Monitoring Authority Statement of GLP Compliance 

FIGURES 

Figure 1..........Linearity 

Figure 2..........Chromatogram of Calibration Solution (100 mg/L) 

Figure 3..........Chromatogram of Biological Control Sample 

Figure 4...........Chromatogram of Test Sample (100 mg/L) REFERENCES 

HAMILTON, M A, RUSSO, R C AND THURSTON, R V (1977) Trimmed Spearman-Karber Method for Estimating Median Lethal Concentration in Toxicity Bioassays. 

Environ Sci Technol 11, 714-719. 

Attached document Remarks 

Appendices - Daphnia sp., 48-Hour Acute Immobilization Test, Benzenesulfonyl chloride 41201583.pdf / 1.05 MB (application/pdf) Appendices 1-8 

Figures 1-4 Daphnia sp., 48-Hour Acute Immobilization Test, Benzenesulfonyl chloride 41201583.pdf / 40.11 KB (application/pdf) Figures 1- 4 

yes 

CONCLUSION 

The acute toxicity of the degradation product to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of greater than 100 mg/L. 

The No Observed Effect Concentration was 100 mg/L. 

SUMMARY 

Introduction 

A study was performed to assess the acute toxicity of the test item toDaphnia magna. The method followed was designed to be compatible with the US EPA Draft 

Ecological Effects Test Guidelines OPPTS 850.1010 (April 1996), ASTM E729-96 and the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphniasp., 

Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008. 

 

 

Methods 

Following a preliminary range-finding test and initial experiment, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at 

nominal concentrations of 32, 56 and 100 mg/L for 48 hours at a temperature of 20°C to 21°C under static test conditions. Due to the extremely acidic nature of the test 

item, the pH of the test preparations was adjusted to 8.0 prior to the addition of the daphnids at the start of the test. The number of immobilizedDaphniawere recorded 

after 24 and 48 hours. 

 

Due to the high water solubility of the test item and the stable nature of the hydrolysis product the use of a flow-through system of preparation was considered 

inappropriate. 

 

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 preparation at 0 and 48 hours showed measured hydrolysis product concentrations of 109 mg/L and 103 mg/L respectively 

were obtained. 

 

Exposure ofDaphnia magnato the degradation product gave EC50values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. 

Endpoint study record: Short-term toxicity to aquatic invertebrates Daphnia sp 48-Ho... Page 8 of 8

file://G:\IUCLIDFiles\Lanxess\Benzenesulfonyl chloride\Lanxess - Benzenesulfonyl ... 21/03/2013