# Endpoint study record: Short

- term toxicity to aquatic invertebrates Daphnia sp

48

- Hour Acute Immobilization Test Benzenesulfonyl chlo ride 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 def ined 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 Experimen tal 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 gui delines and/or minor methodological deficiencies, which do not affect th e 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 Im mobilisation Test) no

according to EU Method C.2 (Acute Toxicity for Daphni a) 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 An alysis for the test item supplied by the Sponsor is given in the attached Appe ndix 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 analys is.

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

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### Details on analytical methods

Vehicle

Details on test solutions The method of analysis, recovery and test preparati on analyses are described in the attched Appendix 5.

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 degradation 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 g ave a chromatographic profile consisting of a singl e 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 wat er 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, Fishe r Scientific, 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 stoc k solution

was further diluted with water to obtain a nominal 100 mg/L calibration standard. A duplicate calibrati on standard was similarly prepared at 100 mg/L. The se duplicate

calibration standards were used to determine the re covery 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 v olumes of

this stock solution were diluted with water to obta in standards in the range of 1.0 to 150 mg/L. A seco nd standard was similarly prepared at a nominal con centration 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 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 stocksolut ion was further diluted with tetrahydrofuran to produce another sol ution of 100 mg/L. Defined volumes of both of these stock solutions were diluted with test medium to o btain spiked recovery samples at concentrations of 1.0 mg/L and 100 mg/L. Five replicates at each concentration lev elwere 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 sonica tion, and then analyzed directly using the instrume ntal 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 a mmonium hydroxide (buffered to pH 7) (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 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 e xperiment were determined by a preliminary range-fi nding test.

In the range-finding test Daphnia magna were expose d to a series of nominal test concentrations of 0.1 0, 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 reco nstituted water and the volume adjusted to 500 mL t o give the 100 mg/L test concentration from which a series of dilutions was made to give f urther test concentrations of 10, 1.0 and 0.10 mg/L

.

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

In the range-finding test 10 daphnids were placed i n 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 transitio n periods. Each 250 mL test and control vessel conta ined 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 co nditions but not exposed to the test item.

A sample of each test concentration was taken for ch emical analysis at 0 and 48 hours in order to deter mine 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 f ollowing 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 rec onstituted water and the volume adjusted to 2.5 lit ers to give the 100 mg/L test concentration from which a series of dilutions was made to give f urther test concentrations of 56, 32, 18 and 10 mg/ L.

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

Exposure Conditions In the initial experiment 250 mL glass jars contain ing approximately 200 mL of test preparation were u sed. At the start of the test 5 daphnids were placed in each test and control vessel at random, i n the test preparations. Four replicate test and co ntrol vessels were prepared. The test vessels were then covered to reduce evaporation and maintai ned 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 minut e dawn and dusk transition periods. The daphnids we re not individually identified, received no food du ring exposure

and the test vessels were not aerated.

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

The test preparations were not renewed during the e xposure period. Any immobilization or adverse reacti ons to exposure were recorded at 24 and 48 hours af ter 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 secon ds after gentle agitation.

Physico-Chemical Measurements Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concen trations and pH were recorded at the start and termination of the test. The pH and dissolved o xygen concentration were measured using a Hach HQ30 d 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 ra nge of pH 7.2 – 7.4 at 10 mg/L through to pH 2.8 – 2.9 at 100 mg/L. It was therefore consider ed that the immobilization observed at the test con centrations of 56 and 100 mg/L may have

been due to the acidic nature of the test item rath er than its true toxicity. As such it was considered appropriate to perform the definitive test using p H 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 a nd the stable nature of the hydrolysis product the use of a flow-through system of preparation was con sidered inappropriate.

Based on the results of the initial experiment the f ollowing 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 ite m was dissolved directly in reconstituted water.

An amount of test item (200 mg) was dissolved in rec onstituted water and the volume adjusted to 2 liter s to give the 100 mg/L test concentration from which a series of dilutions was made to give f urther test concentrations of 56 and 32 mg/L. The p H of each of the test preparations was adjusted to pH 8.0 prior to the addition of the dap hnids.

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

Daphnia magna

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

Adult Daphnia were maintained in 150 mL glass beaker s containing Elendt M7 medium (see attached Appendix 2) in a temperature controlled room at approximatel y 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minu te dawn and dusk transition periods. Each culture wa s fed daily with a mixture of algal suspension (Desmodesmus sub spicatus) and Tetramin® flake food suspension. Cult ure conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the da y before initiation of the test, such that the youn g daphnids produced were approximately 24 hours old . These young were removed from the cultures and used for t esting. The diet and diluent water are considered n ot to contain any contaminant that would affect the integrity or outcome of the study.

A positive control (Harlan Study Number: 41203341) us ed potassium dichromate as the reference item. Deta ils 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 theoreti cal total hardness of 250 mg/l as CaCO3.

Temperature was maintained at 20 °C to 21 °C throug hout the test. Due to a technical oversight the temperature in a s ingle vessel was not logged hourly as required by t he Test Guideline. This was considered to have had no

adverse effect on the outcome of the test given tha t temperature measurements taken from each of the i ndividual test vessels over the 48-Hour test period showed that temperature remained within the guideli ne specification.

The reconstituted water had a pH of 7.8 ± 0.2 adjus ted (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 handhe ld meter See attached Appendix 4 for results.

The reconstituted water was aerated until the disso lved oxygen concentration was approximately air-sat uration value. Dissolved oxygen concentrations were recorded at th e 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 f ollowing 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 f ollowing 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, i n the test preparations. Four replicate test and co ntrol vessels were prepared. The test vessels were then covered to reduce evaporation and maintai ned 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 minut e dawn and dusk transition periods. The daphnids we re not individually identified, received no food du ring exposure

and the test vessels were not aerated.

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

The test preparations were not renewed during the e xposure period. Any immobilization or adverse reacti ons to exposure were recorded at 24 and 48 hours af ter 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 secon ds after gentle agitation.

Physico-Chemical Measurements Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concen trations and pH were recorded at the start and termination of the test. The pH and dissolved o xygen concentration were measured using a Hach HQ30 d 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 te st samples a single-point calibration was performed using duplicate calibration standards. The mean peak areas of each calibration standard were corrected to a nominal co ncentration 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 eq uation:

The recovery rate (R) of the spiked recovery sample s was calculated using the following equation: X = R spl . N ÷ R cal std (1)

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

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

R cal std: mean peak area response for the calibration standar d, 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 tes t sample [mg/L]

x: concentration of the degradation product in 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 ÷ c fort . 100 (3)

where R: recovery rate [%]

c: determined concentration of the degradation product in the spiked recovery sample [mg/L]

c fort : fortified concentration of the degradation product in 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-fi nding test are given in Table 1.

No immobilization was observed at the test concentr ations of 0.10, 1.0 and 10 mg/L. However, immobiliz ation 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 init ial experiment.

Chemical analysis of the 10 and 100 mg/L test prepa rations 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 h ours. These results indicated that the degradation product was stable o ver the test period.

Initial Experiment Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definiti ve 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 ra nge of pH 7.2 – 7.4 at 10 mg/L through to pH 2.8 – 2.9 at 100 mg/L. It was therefore consider ed that the immobilization observed at the test con centrations of 56 and 100 mg/L may have

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

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

Based on the results of the initial experiment the f ollowing test concentrations were assigned to the d efinitive 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 definiti ve test are given in Table 3.

Whilst a single daphnid was observed to be immobili zed in each of the test preparations this was consi dered to be due to natural causes rather than a tru e toxic effect given that less than 10% immobilization occurred. T he 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 col orless solutions throughout the duration of the tes t.

Physico-Chemical Measurements The results of the physico-chemical measurements ar e 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 s ingle vessel was not logged hourly as required by t he Test Guideline. This was considered to have had no adverse effect on the outcome of the test given that temper ature measurements taken from each of the individua l test vessels over the 48-Hour test period showed that temperature remaine d within the guideline specification.

The oxygen concentration in some of the test vessel s was observed to have an air saturation value (ASV) i n excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media su per-saturating the diluent and was considered not t o have had an impact on the outcome or integrity of the test as no adverse effe cts were observed .

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 Sub stances and Mixtures (2000) as the half life was le ss than 1-Hour, analysis of the test preparations was performed for the hydrolysis produ ct only. Analysis of the 100 mg/L test preparation a t 0 and 48 hours (see attached Appendix 5) showed me asured hydrolysis product concentrations of 109 mg/L and 1 03 mg/L respectively were obtained.

Positive Control A positive control (Harlan Study Number: 41203341) us ed potassium dichromate as the reference item at co ncentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

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

Analysis of the immobilization data by the geometric mean method at 24 hours and the trimmed Spearman-Kar ber method (Hamilton et al 1977) at 48 hoursbased on the nominal test concentrations ga ve 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 4 8 hours were 0.56 and 0.32 mg/L respectively. The N o Observed Effect Concentration is based

upon zero immobilization at this concentration.

The results from the positive control with potassiu m 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-Kar ber method (Hamilton et al 1977) at

48 hoursbased on the nominal test concentrations.

TABLES

Table1 Cumulative Immobilization Data in the Ra nge-finding Test 

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...Table 2 Cumulative Immobilization Data in the I nitial Experiment

R 1 – R 4 = Replicates 1 to 4

Table 3 Cumulative Immobilization Data in the D efinitive Test

R 1 – R 4 = Replicates 1 to 4

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

ANALYTICAL TABLES

TABLES Table 1 Preparation of Test Samples

- 

### = not applicable

Table 2 Linearity Data

Nominal

Concentration

(mg/L) Cumulative Immobilized Daphnia

(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 Immobilized Daphnia

(Initial Population: 5 Per Replicate)

24 Hours 48 Hours

R 1 R 2 R 3 R 4 Total % R 1 R 2 R 3 R 4 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 Immobilized Daphnia

(Initial Population: 5 Per Replicate)

24 Hours 48 Hours

R 1 R 2 R 3 R 4 Total % R 1 R 2 R 3 R 4 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 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 

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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

c nom 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

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] [%] [%]

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

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

100 109 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 D efinitive Test Appendix 8....Monitoring Authority Statement of GLP Com pliance

FIGURES Figure 1..........Linearity Figure 2..........Chromatogram of Calibration Soluti on (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 C oncentration in Toxicity Bioassays. Environ Sci Technol 11, 714-719.

Attached document Remarks

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

Figures 1-4 Daphnia sp., 48-Hour Acute Immobilizatio n Test, Benzenesulfonyl chloride 41201583.pdf / 40.1 1 KB (application/pdf) Figures 1- 4

yes

CONCLUSION The acute toxicity of the degradation product to th e freshwater invertebrate Daphnia magna has been in vestigated and gave a 48-Hour EC50 of greater than 1 00 mg/L.

The No Observed Effect Concentration was 100 mg/L.

SUMMARY

Introduction A study was performed to assess the acute toxicity o f the test item to Daphnia 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 Testin g of Chemicals (April 2004) No 202, " Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods Following a preliminary range-finding test and init ial 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 4 8 hours at a temperature of 20°C to 21°C under stat ic test conditions. Due to the extremely acidic nat ure 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 immobilized Daphnia were recorded

after 24 and 48 hours.

Due to the high water solubility of the test item a nd the stable nature of the hydrolysis product the use of a flow-through system of preparation was con sidered inappropriate.

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 D ocument on Aquatic Testing of Difficult Substances and Mixtur es (2000) as the half life was less than 1-Hour, an alysis of the test preparations was performed for t he hydrolysis product only. Analysis of the 100 mg/L test preparat ion at 0 and 48 hours showed measured hydrolysis pr oduct concentrations of 109 mg/L and 103 mg/L respe ctively

were obtained.

Exposure of Daphnia magna to the degradation product gave EC 50 values of greater than 100 mg/L. The No Observed Eff ect Concentration was 100 mg/L. 

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