Sodium 3-(benzothiazol-2-ylthio)propanesulphonate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-21 - 2017-12-01 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 201, adopted 23. Mar. 2006, Annex 5 corrected: 28 July 2011 “Freshwater Alga and Cyanobacteria, Growth Inhibition Test”

Deviations:

yes

Remarks:

not affecting the validity of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Regulation EU No. 2016/266 amending Regulation EC No. 440/2008, Annex IV, Method C.3: “FRESHWATER ALGAE AND CYANOBACTERIA, GROWTH INHIBI-TION TEST,” adopted 07. December 2015

Deviations:

yes

Remarks:

not affecting the validity of the study

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-(benzothiazol-2-ylthio)propanesulphonate

EC Number:

256-401-8

EC Name:

Sodium 3-(benzothiazol-2-ylthio)propanesulphonate

Cas Number:

49625-94-7

Molecular formula:

C10H11NO3S3.Na

IUPAC Name:

sodium 3-(1,3-benzothiazol-2-ylsulfanyl)propane-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

yellow powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
The test item was stored in a closed vessel dark and dry at room temperature (20 ± 5°C).

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 100 mg/L
- Sample storage conditions before analysis: no

Test solutions

Vehicle:

no

Remarks:

a stock solution containing 100 mg/L test item in algal medium was prepared

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A stock solution containing 100 mg/L test item in algal medium (demineralised water enriched with minerals but without algae) was prepared.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Strain:
Genus: Desmodesmus
Species: subspicatus
SAG Strain Number: 86.81
Taxonomic position: Chlorophyta - Chlorophyceae
- Source (laboratory, culture collection): The culture of Desmodesmus subspicatus was obtained in January 2016 by MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: The algae are kept as stock culture on solid agar at 2 - 8 °C. From the stock culture, a permanent culture was prepared. From an aliquot of the permanent culture, the pre-culture was prepared.

ACCLIMATION
- Any deformed or abnormal cells observed: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Test temperature:

21.6 – 22.6 °C

pH:

7.7 - 7.9

Nominal and measured concentrations:

Nominal: 0, 100 mg/L
Measured: n.d., 100.96 mg/L (after 72h)

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test vessels (glass flasks total volume 65 mL) were filled with 45 ± 1 mL of the respective test solution and incubated open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an orbital shaker to keep the algae in suspension.
- Renewal rate of test solution (frequency/flow rate): none
- Initial cells density: 2.0 *10E3 cells/mL
- Control end cells density: 261700 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 5000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (daily)

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: In the pre-test, no inhibition at 100 mg/L was observed

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Any stimulation of growth found in any treatment: Inhibition was found to be -1.54 (Growth Rate (0-72h)) and -7.65 (Yield (0-72h)). Negative inhibition values indicate a stimulation of algal growth compared to the blank control.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
72h ErC50 = 0.94 mg/L (0.90 - 0.98 mg/L)
72h EyC50 = 0.42 mg/L (0.39 - 0.45 mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of ZPS towards algae.
The study was performed as a limit test at the concentration 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.
No significant inhibition of algal growth was observed.
At the start and the end of the test, the content of the test item should be determined using HPLC. Because of error in sequence programming, the test solution was analysed only at the end of the test. This can be stated as uncritical because the measured concentration at the end of the test was 100.96 % of the nominal concentration. Therefore, the determination of the results was based on the nominal concentration.
The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the range of the laboratory.
The pH of the blank control should not fluctuate by more than 1.5 units. The change was 0.2 units in the blank control.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid
As no significant inhibition was observed at the limit dose of 100 mg/l, the EC50 for both growth rate and yield were >100 mg/l, and based on the results of this study, ZPS does not need to be classified as hazardous to the environment, neither acute nor chronic.

Executive summary:

The study was conducted under GLP according to OECD guideline 201 and EU method C.3 on the registered substance itself.

One valid experiment was performed.

The study was performed as a limit test at the concentration 100 mg/L. Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the cell numbers every 24 hours with an electronic particle counter. Growth rate µ and the yield were determined from the cell number at the respective observation times.

No significant inhibition of algal growth was observed.

At the start and the end of the test, the content of the test item should be determined using HPLC. Because of error in sequence programming, the test solution was analysed only at the end of the test. This can be stated as uncritical because the measured concentration at the end of the test was 100.96 % of the nominal concentration. Therefore, the determination of the results was based on the nominal concentration.

The 72h-EC50 values of potassium dichromate (K2Cr2O7, CAS No. 7778-50-9) were determined in a separate reference test. The values lay within the range of the laboratory (growth rate 0.73 - 1.10 mg/L, yield 0.21 – 0.66 mg/L).

The following results for the test item 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) were determined:

 

Table Results of the test item

Endpoint	NOEC	LOEC	EC50
Growth Rate	≥ 100 mg/L	> 100 mg/L	> 100 mg/L
Yield	≥ 100 mg/L	> 100 mg/L	> 100 mg/L

 

ZPS does not need to be classified as hazardous to the environment, neither acute nor chronic.